Note: Descriptions are shown in the official language in which they were submitted.
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COMPOSITIONS AND METHODS FOR INDUCTION OF ANTIGEN-SPECIFIC TOLERANCE
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional Application
Nos. 61/145,941, filed on
January 20, 2009, which application is incorporated herein by reference.
BACKGROUND OF THE INVENTION
[00021 The first step leading to the initiation of an immune response is the
recognition of antigen
fragments presented in association with major histocompatibility complex (MHC)
molecules. Recognition
of antigens can occur directly when the antigens are associated with the MHC
on the surface of foreign
cells or tissues, or indirectly when the antigens are processed and then
associated with the MHC on the
surface of professional antigen presenting cells (APC). Resting T lymphocytes
that recognize such
antigen-MHC complexes become activated via association of these complexes with
the T cell receptor
(Jenkins et al., J. Exp. Med. 165, 302-319, 1987; Mueller et al., J. Immunol.
144, 3701-3709, 1990). A
living organism generally does not display immune response to a self-composing
antigen. This is called
natural or innate immunological tolerance. On the other hand, even if an
antigen is originally
heterogeneous to a living organism, it may not react to the immune response
which is displayed on dosing
of the antigen, depending on when it is dosed, how it is dosed and in what
form it is dosed. This is called
acquired tolerance. If T cells are only stimulated through the T cell
receptor, without receiving an
additional costimulatory signal, they become nonresponsive, anergic, or die,
resulting in downmodulation
of the immune response, and tolerance to the antigen. (Van Gool et al., Eur.
J. Immunol. 29(8):2367-75,
1999; Koenen et al., Blood 95(10):3153-61, 2000). However, if the T cells
receive a second signal, termed
costimulation, T cells are induced to proliferate and become functional
(Lenschow et al., Annu. Rev.
Immunol. 14:233, 1996). The self/non-self recognition is thought to occur at
the interaction level of
antigen presenting cells (e.g. dendritic cells or macrophages), and T
lymphocytes.
[0003] Conventional clinical strategies for general long-term
immunosuppression in disorders associated
with an undesired immune response (e.g., autoimmune disease, graft rejection)
are based on the long-term
administration of broad acting immunosuppressive drugs, for example, signal 1
blockers such as for
example cyclosporin A (CsA), FK506 (tacrolimus) and corticosteroids. Long-term
use of high doses of
these drugs can also have toxic side-effects. Moreover, even in those patients
that are able to tolerate these
drugs, the requirement for life-long immunosuppressive drug therapy carries a
significant risk of severe
side effects, including tumors, serious infections, nephrotoxicity and
metabolic disorders (Penn 2000;
Fishman et al. 1998).
[0004] Methods of inducing antigen-specific tolerance have been developed,
including cell coupling of
an antigen or peptide. For example, in one method, peptide induced cell
coupled tolerance involved
collection, separation and treatment of peripheral blood cells with disease
specific autoantigens and the
ethylene carbodiimide (ECD1) coupling reagent under sterile GMP conditions,
and subsequent re-infusion
into the donor/patient. This process is costly and must be conducted under
closely monitored conditions
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SUBSTITUTE SHEET (RULE 26)
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by skilled practioners and is limited in the number of centers that can
conduct the procedure. The use of
red blood cells as the carrier cell type expands the potential source to
include allogeneic donors thus
increasing the supply of source cells dramatically and potentially expanding
the delivery of this therapy to
any setting certified for blood transfusion. Conventional methods also include
the use of ethylene
carbodiimide fixed autologous splenocytes. These cells express the peptide for
which antigen-specific
tolerance is sought. However, collection and preparation of sufficient numbers
of cells represents a
significant hurdle to the broad utilization of this technology for treating
human autoimmune disease,
transplant rejection and allergic or hyperimmune responses. These approaches
have significant potential
limitations in terms of supply of source cells and necessity for tissue type
matching to minimize immune
response to the carrier. In addition the local treatment of the cells to
couple autoantigens via EDCI
presents a significant quality control issue.
SUMMARY OF THE INVENTION
[0005] Achievement of immune tolerance is desirable in certain instances, for
example, autoimmune
disease, transplant rejection and allergic or hyperimmune responses.
Accordingly, there is a need for
improved approaches that are capable of efficiently inducing long-term immune
tolerance without the
need for administration of high initial doses of immunosuppressive drugs, or
the use of biological material
as a carrier.
[0006] In one embodiment, the present invention provides a composition for
induction of antigen-
specific tolerance comprising a carrier particle attached thereto an apoptotic
signaling molecule and an
antigenic peptide. In another embodiment, the present invention provides a
composition for induction of
antigen-specific tolerance comprising a carrier particle attached thereto an
antigenic peptide. In a
preferred embodiment, the carrier particle is a polystyrene particle. In one
aspect, the composition induces
antigen-specific tolerance in a subject. Where desired, the antigenic peptide
can be an autoimmune
antigen, a transplantation antigen or an allergen. For instance, the antigenic
peptide is myelin basic
protein, acetylcholine receptor, endogenous antigen, myelin oligodendrocyte
glycoprotein, pancreatic
beta-cell antigen, insulin, glutamic acid decarboxylase (GAD), collagen type
11, human carticlage gp39,
fp 130-RAPS, proteolipid protein, fibrillarin, small nucleolar protein,
thyroid stimulating factor receptor,
histones, glycoprotein gp70, pyruvate dehydrogenase dehyrolipoamide
acetyltransferase (PCD-E2), hair
follicle antigen or human tropomyosin isoform 5. In another aspect, the
antigenic peptide is coupled to
the carrier by a conjugate molecule. In yet another aspect, the apoptotic
signaling molecule is a scavenger
receptor ligand such as annexin-1, annexin-5, phosphatidyl serine,
cholesterol, milk fat globule-EGF-
factor 8 (MFG-E8), or Fas-ligand. Where desired, the antigenic peptide can be
fused to the apoptotic
signalling molecule. In yet another aspect, the carrier comprises a quantum
dot. In some instances, the
carrier is a dendrimer, liposome or micelle. The carrier can also be a
nanoparticle or microparticle is less
than 1000 microns in diameter. The nanoparticle or ricroparticle can be
biodegradable.
[0007] The present invention also provides a method of reducing an antigen-
specific immune response in
a subject. The method involves the step of administering to said subject a
composition for induction of
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antigen-specific tolerance. In one embodiment, the composition comprises a
carrier particle attached
thereto an apoptotic signaling molecule and an antigenic peptide, wherein said
composition reduces an
antigen-specific immune response in a subject. In another embodiment, the
composition comprises a
carrier particle attached thereto an antigenic peptide, wherein said
composition reduces an antigen-
specific immune response in a subject. Ina preferred embodiment, the carrier
particle is a polystyrene
particle. Where desired, the antigenic peptide utilized in the subject method
is an autoimmune antigen, a
transplantation antigen, or an allergen. In some instances, the autoimmune
antigen can be one to which
the subject mounts an immune response.
[0008] The present invention further provides a method of treating a subject
having an autoimmune
disorder comprising administering to the subject a composition comprising a
nanoparticle or
microparticle. The nanoparticle or microparticle comprise (a) an inherent or
added apoptotic signaling
molecule; and (b) a pathogenic antigen.
[0009] The present invention also provides a method for ameliorating a
demyelinating disorder in a
subject in need of utilizing any of the composition disclosed herein.
[0010] Further provided in the present invention is a kit for inducing antigen
specific tolerance
comprising: (a) a carrier particle; and (b) an antigenic peptide bound to the
carrier particle.
INCORPORATION BY REFERENCE
[0011] All publications and patent applications mentioned in this
specification are herein incorporated by
reference to the same extent as if each individual publication or patent
application was specifically and
individually indicated to be incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] The novel features of the invention are set forth with particularity in
the appended claims. A
better understanding of the features and advantages of the present invention
will be obtained by reference
to the following detailed description that sets forth illustrative
embodiments, in which the principles of the
invention are utilized, and the accompanying drawings of which:
[0013] Figure 1 depicts the mean clinical score for clinical signs of EAE
following treatment using
peptides conjugated to an artificial carrier. The graph shows that polystyrene
microspheres coupled with
the PLP peptide provided protection from PLP139.151/CFA induced EAE and
abrogated the relapse of
active EAE in SJL mice.
[0014] Figure 2 depicts the effect of administration of peptide-coupled
polystyrene microspheres either
prior to, or after induction of PLP139-151 induced EAE in mice. (A) Pre-
treatment with peptide-coupled
microspheres prior to priming with PLP139_151 + Complete Freund's Adjuvant
(CFA); (B) Pre-treatment
with peptide-coupled microspheres prior to priming with PLP178-191 + Complete
Freund's Adjuvant
(CFA); (C) Post-treatment with peptide-coupled microspheres following priming
with PLP139_151 +
Complete Freund's Adjuvant (CFA).
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[0015] Figure 3 depicts the effect of administration of peptide-coupled
polystyrene microspheres on
delayed type-hypersensitivity ear swelling in a mouse model.
[0016] Figure 4 depicts the effect of administration of peptide-coupled
polystyrene microspheres on CNS
infiltration of leukocytes into the CNS. Leukocyte markers were assayed from
spinal cord slices and
stained for: (A) cellularity; (B) CD4+CD3+ cells, and (C) Foxp3+ cells.
[0017] Figure 5 depicts the effect of administration of peptide-coupled
polystyrene microspheres on
splenectomized mice.
DETAILED DESCRIPTION OF THE INVENTION
[0018] Methods for induction of antigen-specific tolerance are desirable to
prevent or diminish immune
reactions in several instances, including treatment of autoimmune disease,
transplant rejection and allergic
or hyperimmune responses. The present invention utilizes a carrier to present
antigenic peptides and
proteins to the immune system in such a way as to induce antigen-specific
tolerance. Antigen presenting
cells, such as dendritic cells and macrophages, ordinarily trigger an immune
system cascade, but these
same cells are capable of inducing tolerance when the antigen is presented in
the absence of co-
stimulatory molecules and/or the secretion of inflammatory cytokines
(Duperrier, K. et al.
"Immunosuppressive agents mediate reduced allostimulatory properties of
myeloid-derived dendritic cells
despite induction of divergent molecular phenotypes". Mol Immunol 42 (2005),
1531-40; Piemonti, L. et
al. "Glucocorticoids affect human dendritic cell differentiation and
maturation". J Immunol 162 (1999),
6473-81). The carrier of the present invention maybe bound to an antigen
conjugated to a substance (e.g.
ethylene carbodiimide or ECDI) which would allow the particle to be perceived
as a self antigen by an
antigen presenting cell (APC) of the host reticuloendothelial system or
directly by T-cells and allow
presentation of the associated antigens in a tolerance-inducing manner.
Without being bound by theory,
this tolerance may occur by presentation of antigen without associated
upregulation of molecules involved
in immune cell stimulation (for example, MHC class 1/II or costimulatory
molecules).
[0019] In some embodiments, an inert carrier, such as those described below,
are effective to induce
antigen-specific tolerance and/or prevent the onset of an immune related
disease (such as EAE in a mouse
model) and/or diminish the severity of a pre-existing immune related disease.
In some embodiments, the
compositions and methods of the present invention can cause T cells to
undertake early events associated
with T-cell activation, but do not allow T-cells to acquire effector function.
For example, administration
of compositions of the present invention can result in T-cells having a quasi-
activated phenotype, such as
CD69 and/or CD44 upregulation, but do not display effector function, such as
indicated by a lack of IFN-y
or IL-17 synthesis. In some embodiments, administration of compositions of the
present invention results
in T-cells having a quasi-activated phenotype without having conversion of
naive antigen-specific T-cells
to a regulatory phenotype, such as those having CD25+/Foxp3+ phenotypes.
[0020] In some instances, the carrier is further conjugated to a molecule that
mimics a tolerogenic signal.
The addition of an apoptotic signaling molecule is thought to specifically
signal to an APC a non-
dangerous apoptotic uptake signal which indicates to the host that the
associated antigens are self antigens
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and result in a tolerizing response. In other instances, the carrier particle
does not include a separate
apoptotic signaling molecule. Without being bound by theory, the carrier
particle carrying an antigen
specific peptide or protein targets immature B and T cells (e.g., in the
spleen, bone marrow or lymph
nodes) to affect tolerance.
[0021] The invention is useful for treatment of immune related disorders such
as autoimmune disease,
transplant rejection and allergic reactions. Substitution of a synthetic,
biocompatible carrier system
capable of carrying a cellular substrate to induce an antigen-specific
tolerance response could lead to ease
of manufacturing, broad availability of therapeutic agents, increase
uniformity between samples, increase
the number of potential treatment sites and dramatically reduce the potential
for allergic responses to a
carrier cell.
[0022] As used herein, the term "immune response" includes T cell mediated
and/or B cell mediated
immune responses. Exemplary immune responses include T cell responses, e.g.,
cytokine production and
cellular cytotoxicity. In addition, the term immune response includes immune
responses that are indirectly
effected by T cell activation, e.g., antibody production (humoral responses)
and activation of cytokine
responsive cells, e.g., macrophages. Immune cells involved in the immune
response include lymphocytes,
such as B cells and T cells (CD4, CD8+, Thl and Th2 cells); antigen presenting
cells (e.g., professional
antigen presenting cells such as dendritic cells, macrophages, B lymphocytes,
Langerhans cells, and non-
professional antigen presenting cells such as keratinocytes, endothelial
cells, astrocytes, fibroblasts,
oligodendrocytes); natural killer cells; myeloid cells, such as macrophages,
eosinophils, mast cells,
basophils, and granulocytes.
100231 As used herein, the term "anergy," "tolerance," or "antigen-specific
tolerance" refers to
insensitivity of T cells to T cell receptor-mediated stimulation. Such
insensitivity is generally antigen-
specific and persists after exposure to the antigenic peptide has ceased. For
example, anergy in T cells is
characterized by lack of cytokine production, e.g., IL-2. T-cell anergy occurs
when T cells are exposed to
antigen and receive a first signal (a T cell receptor or CD-3 mediated signal)
in the absence of a second
signal (a costimulatory signal). Under these conditions, re-exposure of the
cells to the same antigen (even
if re-exposure occurs in the presence of a costimulatory molecule) results in
failure to produce cytokines
and subsequently failure to proliferate. Thus, a failure to produce cytokines
prevents proliferation.
Anergic T cells can, however, proliferate if cultured with cytokines (e.g., IL-
2). For example, T cell
anergy can also be observed by the lack of IL-2 production by T lymphocytes as
measured by ELISA or
by a proliferation assay using an indicator cell line. Alternatively, a
reporter gene construct can be used.
For example, anergic T cells fail to initiate 1L-2 gene transcription induced
by a heterologous promoter
under the control of the 5' IL-2 gene enhancer or by a multimer of the AP 1
sequence that can be found
within the enhancer (Kang et al. 1992 Science. 257:1134).
[0024] As used herein, the term "immunological tolerance" refers to methods
performed on a proportion
of treated subjects in comparison with untreated subjects where: a) a
decreased level of a specific
immunological response (thought to be mediated at least in part by antigen-
specific effector T
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lymphocytes, B lymphocytes, antibody, or their equivalents); b) a delay in the
onset or progression of a
specific immunological response; or c) a reduced risk of the onset or
progression of a specific
immunological response. "Specific" immunological tolerance occurs when
immunological tolerance is
preferentially invoked against certain antigens in comparison with others.
[0025] Various aspects of the invention are described in further detail in the
following subsections.
Carriers
[0026] The antigen-specific tolerance inducing compositions of the present
invention may be produced
with any of a large variety of carriers including, but not limited to,
particles, beads, branched polymers,
dendrimers, or liposomes. Preferably the carrier is particulate, and generally
spherical, ellipsoidal, rod-
shaped, globular, or polyhedral in shape. Alternatively, however, the carrier
may be of an irregular or
branched shape. In preferred embodiments, the carrier is composed of material
which is biodegradable. It
is further preferred that the carrier have a net neutral or negative charge,
in order to reduce non-specific
binding to cell surfaces which, in general, bear a net negative charge. The
carriers may be capable of
being conjugated, either directly or indirectly, to an antigen to which
tolerance is desired (also referred to
herein as an antigen-specific peptide, antigenic peptide, autoantigen,
inducing antigen or tolerizing
antigen). In some instances, the carrier will have multiple binding sites in
order to have multiple copies
of the antigen-specific peptide exposed and increase the likelihood of a
tolerance response. The carrier
may have one antigenic peptide on the carrier surface or multiple different
antigenic peptides on the
surface. Alternatively, however, the carrier may have a surface to which
conjugating moieties may be
adsorbed without chemical bond formation.
[0027] In some instances, the antigen-specific peptide is delivered to antigen
presenting cells (APCs),
such as dendritic cells (DCs) or macrophages, where lymphocytes are undergoing
maturation (e.g. spleen,
bone marrow, thymus and lymph nodes). There are resident APCs and DCs, for
example, in spleen, bone
marrow, thymus and lymph nodes. Alternatively, the antigen-specific peptide
may be delivered to
peripheral APCs or DCs, where they first internalize the carriers and then
migrate to sites of lymphocyte
maturation (e.g. spleen, bone marrow, thymus or lymph nodes) to activate a
tolerance response. This
generally occurs within 1-3 days. Resident APCs at sites of lymphocyte
maturation may be utilized as
targets.
[0028] The overall size and weight of the carriers are important
considerations. Preferably, the carriers
are microscopic or nanoscopic in size, in order to enhance solubility, avoid
possible complications caused
by aggregation in vivo and to facilitate pinocytosis. Particle size can be a
factor for uptake from the
interstitial space into areas of lymphocyte maturation.
[0029] In various embodiments, the largest cross-sectional diameters of the
composition of the invention
are less than about 1,000 gm, 500 gin, 100 gm, 50 gm, 25 gm, 20 gm, 15 gm, 10
gm, 5 gm, 1 gm, 500
nm, 400 nm, 300 nm, 200 mn or 100 nm. The composition of the present invention
may be chosen to
maximize delivery to lymphocytes, for example, immature lymphocyte such as
those found in the spleen,
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thymus, bone marrow or lymph nodes. In some embodiments, carriers have maximum
diameters of about
5-80 nm. Alternatively, carriers may have maximum diameters of about 10-70 nm,
or 20-60 rim, or 30-50
nm. In some embodiments, the overall weights of the carriers are less than
about 10,000 kDa, less than
about 5,000 kDa, or less than about 1,000, 500, 400, 300, 200 or 100 kDa.
[0030] Preferably the particle surface is composed of a material that
minimizes non-specific or unwanted
biological interactions. Interactions between the particle surface and the
interstitium may be a factor that
plays a role in lymphatic uptake. The particle surface may be coated with a
material to prevent or
decrease non-specific interactions. Steric stabilization by coating particles
with hydrophilic layers such as
poly(ethylene glycol) (PEG) and its copolymers such as PLURONICS (including
copolymers of
poly(ethylene glycol)-bl-poly(propylene glycol)-bl-poly(ethylene glycol)) may
reduce the non-specific
interactions with proteins of the interstitium as demonstrated by improved
lymphatic uptake following
subcutaneous injections. All of these facts point to the significance of the
physical properties of the
particles in terms of lymphatic uptake. Biodegradable polymers may be used to
make all or some of the
polymers and/or particles and/or layers. Biodegradable polymers may undergo
degradation, for example,
by a result of functional groups reacting with the water in the solution. The
term "degradation" as used
herein refers to becoming soluble, either by reduction of molecular weight or
by conversion of
hydrophobic groups to hydrophilic groups. Polymers with ester groups are
generally subject to
spontaneous hydrolysis, e.g., polylactides and polyglycolides. Many peptide
sequences subject to specific
enzymatic attack are known, e.g., as degraded by collagenases or
metalloproteinases: sequences that are
degraded merely by biological free radical mechanisms are not specifically
degraded. Polymers with
functional groups that are oxidation-sensitive will be chemically altered by
mild oxidizing agents, with a
test for the same being enhanced solubilization by exposure to 10% hydrogen
peroxide for 20 h in vitro.
[0031] Carriers of the present invention may also contain additional
components. For example, carriers
may have imaging agents incorporated or conjugated to the carrier. An example
of a carrier nano sphere
having an imaging agent that is currently commercially available is the Kodak
X-sight nanospheres.
Inorganic quantum-confined luminescent nanocrystals, known as quantum dots
(QDs), have emerged as
ideal donors in FRET applications: their high quantum yield and tunable size-
dependent Stokes Shifts
permit different sizes to emit from blue to infrared when excited at a single
ultraviolet wavelength.
(Bruchez, et al., Science, 1998, 281, 2013; Niemeyer, C. M Angew. Chem. Int.
Ed. 2003, 42, 5796;
Waggoner, A. Methods Enzymol. 1995, 246, 362; Brus, L. E. J. Chem. Phys. 1993,
79, 5566). Quantum
dots, such as hybrid organic/inorganic quantum dots based on a class of
polymers known as dendrimers,
may used in biological labeling, imaging, and optical biosensing systems.
(Lemon, et al., J. Am. Chem.
Soc. 2000, 122, 12886). Unlike the traditional synthesis of inorganic quantum
dots, the synthesis of these
hybrid quantum dot nanoparticles does not require high temperatures or highly
toxic, unstable reagents.
(Etienne, et al., Appl. Phys. Lett. 87, 181913, 2005).
Microbead or Nanobead Carriers
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[00321 In some embodiments, the antigen-specific tolerance inducing
compositions of the present
invention comprise a carrier which is a microparticle or nanoparticle. In some
instances, the microparticle
or nanoparticle is a substantially spherical bead or a porous bead.
[00331 Carrier particles can be formed from a wide range of materials. The
particle is preferably
composed of a material suitable for biological use. For example, particles may
be composed of glass,
silica, polyesters of hydroxy carboxylic acids, polyanhydrides of dicarboxylic
acids, or copolymers of
hydroxy carboxylic acids and dicarboxylic acids. More generally, the carrier
particles may be composed
of polyesters of straight chain or branched, substituted or unsubstituted,
saturated or unsaturated, linear or
cross-linked, alkanyl, haloalkyl, thioalkyl, aminoalkyl, aryl, aralkyl,
alkenyl, aralkenyl, heteroaryl, or
alkoxy hydroxy acids, or polyanhydrides of straight chain or branched,
substituted or unsubstituted,
saturated or unsaturated, linear or cross-linked, alkanyl, haloalkyl,
thioalkyl, aminoalkyl, aryl, aralkyl,
alkenyl, aralkenyl, heteroaryl, or alkoxy dicarboxylic acids. Additionally,
carrier particles can be quantum
dots, or composed of quantum dots, such as quantum dot polystyrene particles
(Joumaa et al. (2006)
Langmuir 22:1810-6). Carrier particles including mixtures of ester and
anhydride bonds (e.g., copolymers
of glycolic and sebacic acid) may also be employed. For example, carrier
particles may comprise
materials including polyglycolic acid polymers (PGA), polylactic acid polymers
(PLA), polysebacic acid
polymers (PSA), poly(lactic-co-glycolic) acid copolymers (PLGA), poly(lactic-
co-sebacic) acid
copolymers (PLSA), poly(glycolic-co-sebacic) acid copolymers (PGSA), etc.
Other biocompatible,
biodegradable polymers useful in the present invention include polymers or
copolymers of caprolactones,
carbonates, amides, amino acids, orthoesters, acetals, cyanoacrylates and
degradable urethanes, as well as
copolymers of these with straight chain or branched, substituted or
unsubstituted, alkanyl, haloalkyl,
thioalkyl, aminoalkyl, alkenyl, or aromatic hydroxy- or di-carboxylic acids.
In addition, the biologically
important amino acids with reactive side chain groups, such as lysine,
arginine, aspartic acid, glutamic
acid, serine, threonine, tyrosine and cysteine, or their enantiomers, may be
included in copolymers with
any of the aforementioned materials to provide reactive groups for conjugating
to antigen peptides and
proteins or conjugating moieties. Biodegradable materials suitable for the
present invention include PLA,
PGA, and PLGA polymers. Biocompatible but non-biodegradable materials may also
be used in the
carrier particles of the invention. For example, non-biodegradable polymers of
acrylates, ethylene-vinyl
acetates, acyl substituted cellulose acetates, non-degradable urethanes,
styrenes, vinyl chlorides, vinyl
fluorides, vinyl imidazoles, chlorosulphonated olefins, ethylene oxide, vinyl
alcohols, TEFLON
(DuPont, Wilmington, Del.), and nylons may be employed.
[0034] Suitable beads which are currently available commercially include
polystyrene beads such as
FluoSpheres (Molecular Probes, Eugene, Oreg.).
[0035] In one embodiment, microparticles or nanoparticles are taken up by
APC's. The size of the
microparticle or nanoparticle is preferably in the range to trigger
phagocytosis or pinocytosis in the APC.
In some embodiments, the microparticle or nanoparticle is in a range of about
100nm to 50 m, 1 gm to
40 gm, 5 gm to 30 gm or 10 gm to 20 gm. In some embodiments, the microparticle
or nanoparticle is
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less than 100 gm, 50 gm, 25 gm, 20 gm, 15 gm, 10 gm, 5 gm, 1 gm, 500 nm or 100
rim. In other
embodiments, the microparticle or nanoparticle is more than 10 nm, 50 rim, 100
nm, 500 nm, 600 nm, 700
nm, 800 nm, 900 nm or 1 gm.
[0036] In one embodiment, microparticles and nanoparticles are taken up in
areas having immature
lymphocytes (e.g. spleen, bone marrow, thymus or lymph nodes). As documented
herein, size is related
to carrier uptake and retention in areas having immature lymphocytes. It is
desirable to obtain both
efficient uptake and retention, since carrier properties, such as size and
surface characteristics, can have
conflicting effects. In general, smaller particles have better uptake than
larger particles but lower
retention. Carriers with a size of about 5 nm to about 10 gm diameter are
preferred; artisans will
immediately appreciate that all the ranges and values within the explicitly
stated ranges are contemplated,
e.g., 25nm, 50nm, 100 nm, 200nm, 300nm, 400nm, 500nm, 600nm, 700nm, 800nm,
900nm, 1 m, 2 m,
3gm, 4 m, 5gm4 6 m, 7 m, 8gm, 9 m or 10 m. The nanoparticles may be made in a
collection that of
particles that has a mean diameter from about 5 to about 100 rim; artisans
will immediately appreciate that
all the ranges and values within the explicitly stated ranges are
contemplated, e.g., from about 10 to about
70 nm. The size distribution of such a collection of particles can be
controlled so that a coefficient of
variation (standard deviation divided by mean particle size) around a mean
diameter of a collection of the
particles may be less than about 50, about 35, about 20, about 10, or about 5
nm. A person of skill in the
art will immediately appreciate that all the ranges and values within the
explicitly stated ranges are
contemplated.
[0037] Physical properties are also related to a nanoparticle's usefulness
after uptake and retention in
areas having immature lymphocytes. These include mechanical properties such as
rigidity or rubberiness.
Some embodiments are based on a rubbery core, e.g., a polypropylene sulfide)
(PPS) core with an
overlayer, e.g., a hydrophilic overlayer, as in PEG, as in the PPS-PEG system
recently developed and
characterized for systemic (but not targeted or immune) delivery. The rubbery
core is in contrast to a
substantially rigid core as in a polystyrene or metal nanoparticle system. The
term rubbery refers to
certain resilient materials besides natural or synthetic rubbers, with rubbery
being a term familiar to those
in the polymer arts. For example, cross-linked PPS can be used to form a
hydrophobic rubbery core. PPS
is a polymer that degrades under oxidative conditions to polysulfoxide and
finally polysulfone,
transitioning from a hydrophobic rubber to a hydrophilic, water-soluble
polymer. Other sulfide polymers
may be adapted for use, with the term sulfide polymer referring to a polymer
with a sulfur in the backbone
of the mer. Other rubbery polymers that may be used are polyesters with glass
transition temperature
under hydrated conditions that is less than about 37 C. A hydrophobic core can
be advantageously used
with a hydrophilic overlayer since the core and overlayer will tend not to
mingle, so that the overlayer
tends to stericly expand away from the core. A core refers to a particle that
has a layer on it. A layer refers
to a material covering at least a portion of the core. A layer may be adsorbed
or covalently bound. A
particle or core may be solid or hollow. Rubbery hydrophobic cores are
advantageous over rigid
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hydrophobic cores, such as crystalline or glassy (as in the case of
polystyrene) cores, in that higher
loadings of hydrophobic drugs can be carried by the particles with the rubbery
hydrophobic cores.
[0038] Another physical property is the surface's hydrophilicity. A
hydrophilic material may have a
solubility in water of at least 1 gram per liter when it is uncrosslinked.
Steric stabilization of particles with
hydrophilic polymers can improve uptake from the interstitium by reducing non-
specific interactions;
however, the particles' increased stealth nature can also reduce
internalization by phagocytic cells in areas
having immature lymphocytes. The challenge of balancing these competing
features has been met,
however, and this application documents the creation of nanoparticles for
effective lymphatic delivery to
DCs and other APCs in lymph nodes. Some embodiments include a hydrophilic
component, e.g., a layer
of hydrophilic material. Examples of suitable hydrophilic materials are one or
more of polyalkylene
oxides, polyethylene oxides, polysaccharides, polyacrylic acids, and
polyethers. The molecular weight of
polymers in a layer can be adjusted to provide a useful degree of steric
hindrance in vivo, e.g., from about
1,000 to about 100,000 or even more; artisans will immediately appreciate that
all the ranges and values
within the explicitly stated ranges are contemplated, e.g., between 10,000 and
50,000.
[0039] The nanoparticles may incorporate functional groups for further
reaction. Functional groups for
further reaction include electrophiles or nucleophiles; these are convenient
for reacting with other
molecules. Examples of nucleophiles are primary amines, thiols, and hydroxyls.
Examples of electrophiles
are succinimidyl esters, aldehydes, isocyanates, and maleimides.
[0040] In preferred embodiments, carrier beads are employed having an average
diameter of about 5-
1000nm, 10-400 nm, 20-200 nm, 30-100nm or about 40-50 nm.
[0041] Antigen-specific tolerance may be induced through use of microparticles
or nanoparticles as
described which are bound to one or more antigenic peptides.
[0042] In one series of embodiments, the present invention provides
compositions and methods for the
induction of tolerance using antigenic peptide attached to a nanoparticle
carrier particle. In some
instances, the carrier also contains an apoptotic signaling molecule. The
carrier particle may be solid,
hollow, or porous.
Polystyrene beads
[0043] Polystyrene beads have been found to elicit an antigen-specific
tolerance effect for antigenic
peptides bound to the surface without the need for an apoptotic signaling
molecule. In one embodiment,
the invention provides crosslinked, functionalized polystyrene beads, having
excellent properties, such as
exceptional uniformity in bead size distribution, pore size, density, swelling
properties and/or tolerance to
solvents and reagents typically used in oligomer synthesis. In some preferred
embodiments, the beads
have superior loading characteristics. In some preferred embodiments, the
beads have a loading capability
of at least about 50 tmole per gram of bead; of at least about 100 .tmole per
gram of bead; of at least
about 150 mole per gram of bead; of at least about 200 .tmole per gram of
bead; of at least about 250
pmole per gram of bead; of at least about 300 pmole per gram of bead; of at
least about 350 mole per
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gram of bead; of at least about 400 gmole per gram of bead; or at least about
450 gmole per gram of bead.
In some embodiments, the bead has a loading capability of from about 100 pmole
per gram of bead to
about 350 gmole per gram of bead.
[0044] Polystyrene particles in the range of 40-50 nm are known to trigger a
danger signal recognized by
dendritic cells (DCs). Polystyrene beads are known to accumulate in lymph
nodes at intermediate sizes
(40 nm) more than smaller (20 nm) and larger (>100 mn) sizes. In some
instances, polystyrene particles
between 10-100nm, 20-80nm, 30-70nm, or 40-50nm may be desired.
[0045] The polystyrene bead size may be important in triggering a signal for
DCs because DCs have
evolved to recognize viral size ranges. The bead size, therefore, would
control successful DC targeting,
with correctly sized beads being recognized by DCs in the periphery.
Additionally, the structure of the
spleen lends itself to uptake of the particles by macrophages.
[0046] In various embodiments, the largest cross-sectional diameters of the
composition of the invention
are less than about 1,000 gm, 500 gm, 100 gm, 50 gm, 25 gm, 20 gm, 15 gm, 10
gm, 5 gm, 1 gm, 500
mn, 400 nm, 300 nm, 200 rim or 100 nm. The composition of the present
invention may be chosen to
maximize delivery to lymphocytes, for example, immature lymphocyte such as
those found in the spleen,
thymus, bone marrow or lymph nodes. In some embodiments, carriers have maximum
diameters of about
10-500 nm. Alternatively, carriers may have maximum diameters of about 100-500
nm, or 250-500 nm, or
300-500 nm. In some embodiments, the overall weights of the carriers are less
than about 10,000 kDa,
less than about 5,000 kDa, or less than about 1,000, 500, 400, 300, 200 or 100
kDa.
[0047] In one series of embodiments, the present invention provides
compositions and methods for the
induction of tolerance using antigenic peptide attached to a polystyrene bead.
In some instances, the
antigenic peptide is linked to the polystyrene bead via the N-terminus of the
antigenic peptide to the
carboxyl sites on the polystyrene beads. In some instances, the carrier also
contains an apoptotic signaling
molecule.
Branched Polymer Carriers/Dendrimers
[0048] In some embodiments, the tolerance inducing compositions of the present
invention comprises a
carrier which is a branched polymer, such as a dendrimer. Branched polymers
have numerous chain-ends
or termini which can be functionalized and, therefore, can be conjugated to a
multiplicity of tolerance
inducing complexes, either directly or indirectly through conjugating
moieties.
[0049] Some polymer systems are themselves nanoparticulate and are included in
the term nanoparticle.
For example, dendrimers are a class of polymer that can be nanoparticulate in
the nm range. These
polymers comprise a high number of functional groups at their surface, for
example which have been used
to conjugate to biomolecules and other groups. Analogously, antigen could be
conjugated to the
dendrimer surface. Moreover, the functional groups on the dendrimer surface
could be optimized for
complement activation, for example by hydroxylation. Some dendrimer-DNA
complexes have been
demonstrated to activate complement. Dendrimers represent an interesting
nanoparticulate chemistry that
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could be adapted for lymphatic targeting using the techniques described
herein, for antigen conjugation,
and for complement activation, e.g., as in U.S. Pat. Pub. Nos. 2004/0086479,
2006/0204443, and in U.S.
Pat. Nos. 6,455,071 and 6,998,115, which are hereby incorporated by reference
herein to the extent they
do not contradict what is explicitly disclosed.
[0050] On the other hand, dendrimers have a shape that is highly dependent on
the solubility of its
component polymers in a given environment, and can change dramatically
according to the solvent or
solutes around it, e.g., changes in temperature, pH, ion content, or after
uptake by a DC. In contrast,
nanoparticles that have physical dimensions that are relatively more stable
than dendrimers or other
merely branched polymer systems can be useful for storage purposes or as
related to or biological activity,
e.g., a solid core with a hydrophilic corona will consistently present the
corona to its environment.
Accordingly, some embodiments of nanoparticles rely on particles that are not
dendrimers, or that have a
core that is a solid and/or have a core that is a cross-linked hydrogel. A PPS-
based nanoparticle is not a
dendrimer and has a solid core.
[0051] Dendrimers, also known as arborols, cascade molecules, dendritic
polymers, or fractal polymers,
are highly branched macromolecules in which the branches emanate from a
central core. Dendrimers can
be made from various materials, including, but not limited to, polyamidoamine,
polyamidoalcohol,
polyalkyleneimine such as polypropyleneimine or polyethyleneimine,
polyalkylene such as polystyrene or
polyethylene, polyether, polythioether, polyphosphonium, polysiloxane,
polyamide, polyaryl polymer, or
combinations thereof. Dendrimers have also been prepared from amino acids
(e.g., polylysine).
Preferably, dendrimers are employed which terminate in carboxyl or other
negatively charged reactive
groups in order to facilitate conjugation.
[0052] Dendrimers are known in the art and are chemically defined globular
molecules, generally
prepared by stepwise or reiterative reaction of multifunctional monomers to
obtain a branched structure
(see, e.g., Tomalia et al. (1990) Angew. Chem. Int. Ed. Engl. 29:138-75). A
variety of dendrimers are
known, e.g., amine-terminated polyamidoamine, polyethyleneimine and
polypropyleneimine dendrimers.
Exemplary dendrimers useful in the present invention include "dense star"
polymers or "starburst"
polymers such as those described in U. S. Pat. Nos. 4,587,329; 5,338,532; and
6,177,414, including
poly(amidoamine) dendrimers ("PAMAM"). Still other multimeric spacer molecules
suitable for use
within the present invention include chemically-defined, non-polymeric valency
platform molecules such
as those disclosed in U.S. Pat. No. 5,552,391; and PCT application
publications WO 00/75105, WO
96/40197, WO 97/46251, WO 95/07073, and WO 00/34231. Many other suitable
multivalent spacers can
be used and will be known to those of skill in the art. For example,
dendrimers and their use are described
in US Pat App No. 20070238678, which is hereby incorporated by reference in
its entirety.
[0053] Such dendrimers include but are not limited to polyamidoamine (PAMAM)
dendrimers,
poly(propyleneimine) (PP1) dendrimers, poly(triazine)dendrimers, poly(ether-
hydroxylamine) (PEHAM)
dendrimers, which may have their Z groups modified or selected to force the
chelating agents exclusively
into the dendritic polymer interior or in combination with encapsulation,
allow association with the
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surface of the dendritic polymer. Examples of some such Z surfaces are those
which do not interact with
the ligand; such Z groups are hydroxyl, ester, acid, ether, carboxylic salts,
alkyls, glycols, such as for
example hydroxyl groups especially those from amidoethanol,
amidoethylethanolamine,
tris(hydroxymethyl)amine, carbomethoxypyrrolidinone, amido, thiourea, urea,
carboxylate, succinamic
acid and polyethylene glycol or primary or primary, secondary or tertiary
amine groups with or without
hydroxyl alkyl modifications. Other suitable surface groups may include any
such functionality that would
allow associative attachment (associate with) the dendritic polymer surface
and include but are not limited
to receptor mediated targeting groups (e.g., folic acid, antibodies, antibody
fragments, single chain
antibodies, proteins, peptides, oligomers, oligopeptides, or genetic
materials) or other functionality that
would facilitate biocompatibility, biodistribution, solubility or modulate
toxicity. In a preferred
embodiment, the dendrimers contain amino and/or carboxy binding sites on the
surface.
100541 Suitable dendrimers which are currently available commercially include
polyamidoamine
dendrimers such as StarburstTM dendrimers (Dendritech, Midland, Mich.). The
StarburstTM dendrimers
terminate in either amine groups or carboxymethyl groups which may be used,
with or without further
modification, and with or without interposing conjugating moieties, to
conjugate antigen peptides and
proteins to the surface of these carriers.
100551 In one method of dendrimer production, dendrimers are synthesized
outward from a core
molecule by sequential addition of layers of monomers. The first round of
dendrimer synthesis adds a
single layer or "generation" of monomers to the core, with each monomer having
at least one free, reactive
terminus. Each subsequent round of polymerization results in the expansion of
the dendrimer by one layer
and increases the number of free, reactive termini. This process can be
repeated numerous times to
produce dendrimers of desired diameter or mass. As the density of the branches
increases, the outermost
branches arrange themselves in the form of a sphere surrounding a lower
density core. See, for example,
U.S. Pat. No. 5,338,532, which is hereby incorporated by reference in its
entirety. In addition, by varying
the shape of the core molecules, dendrimers may be produced in rod-shaped,
disk-like, and comb-like
forms. The resulting dendrimers may possess an arbitrarily large number of
free, reactive termini, to
which a multiplicity of antigen peptides and proteins may be conjugated,
either directly or indirectly. In a
preferred embodiment, the dendrimers are spherical or ovoid in shape.
[00561 Dendrimers may vary in weight, size, shape and number of terminal
reactive groups. For example,
dendrimers may range in weight from 100 to 10000 kDa, or 200 to 5000 kDa, or
250 to 2500 kDa.
Dendrimers may also range in size from 20 to 1000 tun, 30 to 500 nrn, or 50 to
250 urn in the longest
dimension.
[00571 The use of dendrimers, e.g., PANAM or PPI dendrimers, enables the
creation of cationic spherical
particles with a specific number of amino binding sites on the surface. The
size of these particles can be
selected to optimize loading and minimize steric hindrance between surface
linked antigen peptides. For
example, in most applications PANAM dendrimers of 6-7 generations have been
used resulting in
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particles of 50 - 125 kDa molecular weights, 60 - 90 angstrom diameter
(roughly similar in size as
hemoglobin, IgG or histones), and 100 - 1500 active surface groups.
[0058] Multivalent spacers with a variety of valencies are useful in the
practice of the invention, and in
various embodiments the multivalent spacer is bound to between about 3 and
about 400 nucleic acid
moieties, often from 3 to 100, sometimes from 3-50, frequently from 3-10, and
sometimes more than 400
nucleic acid moieties. In various embodiments, the multivalent spacer is
conjugated to more than 10, more
than 25, more than 50, or more than 500 nucleic acid moieties (which may be
the same or different). It
will be appreciated that, in certain embodiments comprising a multivalent
spacer, the invention provides a
population with slightly different molecular structures. For example,
dendrimers of the present invention
may be composed of a somewhat heterogeneous mixture of molecules produced,
i.e., comprising different
numbers (within or predominantly within a determinable range) of nucleic acid
moieties joined to each
dendrimer molecule. In a preferred embodiment, the dendrimers are of a similar
size and shape, i.e.,
composed of numbers of nucleic acid moieties that vary within 20%, 15%, 10%,
5%, 2% or 1 % of each
other.
[00591 Non-dendrimer branched polymers may also be employed in the invention,
and may be produced
from the same general classes of materials as dendrimers. The synthesis of
such branched polymers is also
well known in the art.
[00601 Branched polymers may include at least 5 termini, at least 10 termini,
or at least 100 termini.
Branched polymers may include between 5 and 500 termini, preferably between 10
and 400 termini and
more preferably between 50 and 250 termini.
100611 In some embodiments, the tolerance inducing compositions of the present
invention provides for
the production of conjugates wherein a tolerance inducing complex is
conjugated to a branched or linear
polymer.
Liposome Carriers
[00621 In some embodiments, the multimeric antigen peptide or protein
conjugate comprises a carrier
which is a liposome or micelle. Liposomes, also called lipid vesicles, are
aqueous compartments enclosed
by lipid membranes, and are typically formed by suspending a suitable lipid in
an aqueous medium, and
shaking, extruding, or sonicating the mixture to yield a dispersion of
vesicles. Various forms of
liposomes, including unilamellar vesicles and multilamellar vesicles, may be
used in the present
invention.
[00631 Micellar systems may also display the same useful characteristics as
described above, including
micelles formed from AB and ABA block copolymers of poly(ethylene glycol) and
PPS. When such
copolymers are formed with a molecular fraction of poly(ethylene glycol) that
is relatively high, e.g., in
excess of approx. 40%, then spherical micelles can be expected to form under
certain conditions. These
micelles can be small, e.g., meeting the size mentioned above for lymphatic
entry, and may optionally be
grafted with an overlayer of PEG, or otherwise incorporate PEG or other
polymers to achieve similar
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properties. Moreover, they can be conjugated with antigen, as taught herein,
danger signals or both at the
micelle surface. The block copolymer can terminate in a hydroxyl group, for
complement activation, and
it is particularly beneficial to have the hydrophilic block terminate in a
hydroxyl group, so that this
hydroxyl group will be more readily available on the micellar nanoparticle
surface for complement
binding. Such hydroxylated such surfaces can be tailored to effectively
activate complement. A
particularly useful hydrophilic block is PEG, terminated in a hydroxyl group.
In addition to micelle-
forming polymer architectures, block sizes and block size ratios can be
selected to form vesicular
structures. There also exists a number of other possible chemical compositions
of micellar formulations
that may be used.
[0064] In another series of embodiments, the present invention provides for
the production of multimeric
antigen peptide and protein conjugates in which a multiplicity of antigen
peptides and proteins are
conjugated to the outer surface of a liposome.
[0065] Liposomes may be prepared from a variety of lipid materials including,
but not limited to, lipids
of phosphatidyl choline, phosphatidyl serine, phosphatidyl inositol,
phosphatidyl glycerol, phosphatidyl
ethanolamine, phosphatidic acid, dicetyl phosphate, mono sialoganglioside,
polyethylene glycol, stearyl
armine, ovolecithin and cholesterol, as well as mixtures of these in varying
stoichiometries. Liposomes, as
used herein, may also be formed from non-lipid amphipathic molecules, such as
block copolymers of
poly(oxyethylene-b-isoprene-b-oxye- thylene) and the like. In preferred
embodiments, the liposomes are
preparedfrom lipids that will form negatively charged liposomes, such as those
produced from
phosphatidyl serine, dicetyl phosphate, and dimyristoyl phosphatidic acid.
[0066] The surfaces of liposomes may also be modified to reduce immunogenicity
or to provide
convenient reactive groups for conjugation. For example, sialic acid or other
carbohydrates, or
polyethylene glycol or other alkyl or alkenyl polymers, may be attached to the
surface of a liposome to
reduce immunogenicity. Alternatively, liposomes may be produced bearing a
conjugating moiety such as
biotin by inclusion of a small molar percentage of, for example, biotin-X-
dipalmitoylphosphatidyle-
thanolamine (Molecular Probes, Eugene, Oreg.) in the liposome.
Means of Conjugating antigen peptides and proteins to a Carrier
[0067] A great variety of means, well known in the art, may be used to
conjugate antigenic peptides and
proteins to carriers. These methods include any standard chemistries which do
not destroy or severely
limit the biological activity of the antigen peptides and proteins, and which
allow for a sufficient number
of antigen peptides and proteins to be conjugated to the carrier in an
orientation which allows for
interaction of the antigen peptide or protein with a cognate T cell receptor.
Generally, methods are
preferred which conjugate the C-terminal regions of an antigen peptide or
protein, or the C-terminal
regions of an antigen peptide or protein fusion protein, to the carrier. The
exact chemistries will, of
course, depend upon the nature of the carrier material, the presence or
absence of C-terminal fusions to
the antigen peptide or protein, and/or the presence or absence of conjugating
moieties.
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[0068] Functional groups can be located on the particle as needed for
availability. One location can be as
side groups or termini on the core polymer or polymers that are layers on a
core or polymers otherwise
tethered to the particle. For instance, examples are included herein that
describe PEG stabilizing the
nanoparticles that can be readily functionalized for specific cell targeting
or protein and peptide drug
delivery.
[0069] Conjugates such as ethylene carbodiimide (ECDI), hexamethylene
diisocyanate, propyleneglycol
di-glycidylether which contain 2 epoxy residues, and epichlorohydrin may be
used for fixation of peptides
or proteins to the carrier surface. Without being bound by theory, ECDI is
suspected of carrying out two
major functions for induction of tolerance: (a) it chemically couples the
protein/peptides to the cell surface
via catalysis of peptide bond formation between free amino and free carboxyl
groups; and (b) it induces
the carrier to mimic apoptotic cell death such that they are picked up by host
antigen presenting cells in
the spleen and induce tolerance. It is this presentation to host T-cells in a
non-immunogenic fashion that
leads to direct induction of anergy in autoreactive cells. In addition, ECDI
serves as a potent stimulus for
the induction of specific regulatory T cells.
[0070] In one series of embodiments, the antigen peptides and proteins are
bound to the carrier via a
covalent chemical bond. For example, a reactive group or moiety near the C-
terminus of the antigen (e.g.,
the C-terminal carboxyl group, or a hydroxyl, thiol, or amine group from an
amino acid side chain) may
be conjugated directly to a reactive group or moiety on the surface of the
carrier (e.g., a hydroxyl or
carboxyl group of a PLA or PGA polymer, a terminal amine or carboxyl group of
a dendrimer, or a
hydroxyl, carboxyl or phosphate group of a phospholipid) by direct chemical
reaction. Alternatively, there
may be a conjugating moiety which covalently conjugates to both the antigen
peptides and proteins and
the carrier, thereby linking them together.
[0071] Reactive carboxyl groups on the surface of a carrier may be joined to
free amines (e.g., from Lys
residues) on the antigen peptide or protein, by reacting them with, for
example, 1-ethyl-3-[3,9-dimethyl
aminopropyl] carbodiimide hydrochloride (EDC) or N-hydroxysuccinimide ester
(NHS). Similarly, the
same chemistry may be used to conjugate free amines on the surface of a
carrier with free carboxyls (e.g.,
from the C-terminus, or from Asp or Glu residues) on the antigen peptide or
protein. Alternatively, free
amine groups on the surface of a carrier may be covalently bound to antigen
peptides and proteins, or
antigen peptide or protein fusion proteins, using sulfo-SIAB chemistry,
essentially as described by Arano
et al. (1991) Bioconjug. Chem. 2:71-6.
[0072] In another embodiment, a non-covalent bond between a ligand bound to
the antigen peptide or
protein and an anti-ligand attached to the carrier may conjugate the antigen
to the carrier. For example, a
biotin ligase recognition sequence tag may be joined to the C-terminus of an
antigen peptide or protein,
and this tag may be biotinylated by biotin ligase. The biotin may then serve
as a ligand to non-covalently
conjugate the antigen peptide or protein to avidin or streptavidin which is
adsorbed or otherwise bound to
the surface of the carrier as an anti-ligand. Alternatively, if the antigen
peptides and proteins are fused to
an immunoglobulin domain bearing an Fe region, as described above, the Fe
domain may act as a ligand
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and protein A, either covalently or non-covalently bound to the surface of the
carrier, may serve as the
anti-ligand to non-covalently conjugate the antigen peptide or protein to the
carrier. Other means are well
known in the art which may be employed to non-covalently conjugate antigen
peptides and proteins to
carriers, including metal ion chelation techniques (e.g., using a poly-His tag
at the C-terminus of the
antigen peptide or protein or antigen peptide or protein fusion proteins, and
a Ni+-coated carrier), and
these methods may be substituted for those described here.
[0073] Conjugation of a nucleic acid moiety to a platform molecule can be
effected in any number of
ways, typically involving one or more crosslinking agents and functional
groups on the nucleic acid
moiety and platform molecule. Linking groups are added to platforms using
standard synthetic chemistry
techniques. Linking groups can be added to nucleic acid moieties using
standard synthetic techniques.
Apoptosis signaling molecules
[0074] In some embodiments, the tolerance inducing compositions of the present
invention contain an
apoptosis signaling molecule. The apoptotic signaling molecules serve allow a
carrier to be perceived as
an apoptotic body by antigen presenting cells of the host, such as cells of
the host reticuloendothelial
system. This allows presentation of the associated peptide epitopes in a
tolerance-inducing manner.
Without being bound by theory, this is presumed to prevent the upregulation of
molecules involved in
immune cell stimulation, such as MHC class 1/H, and costimulatory molecules.
These apoptosis signaling
molecules may also serve as phagocytic markers. For example, apoptosis
signaling molecules suitable for
the present invention have been described in US Pat App No. 20050113297, which
is hereby incorporated
by reference in its entirety. Molecules suitable for the present invention
include molecules that target
phagocytes, which include macrophages, dendritic cells, monocytes and
neutrophils.
[0075] Molecules suitable as apoptotic signaling molecules act to enhance
tolerance of the associated
peptides. Additionally, a carrier bound to an apoptotic signaling molecule can
be bound by C l q in
apoptotic cell recognition (Paidassi et al., (2008) J. Immunol. 180:2329-
2338). For example, molecules
that may be useful as apoptotic signaling molecules include phosphatidyl
serine, annexin- 1, annexin-5,
milk fat globule-EGF-factor 8 (MFG-E8), or the family of thrombospondins.
[0076] Thrombospondins are a family of extracellular proteins that participate
in cell-to-cell and cell-to-
matrix communication. They regulate cellular phenotype during tissue genesis
and repair. In addition,
thrombospondin-1 (TSP-1) is expressed on apoptotic cells and is involved in
their recognition by
macrophages. Thrombospondin-1 is therefore another phagocytic marker that can
be used to enhance
phagocytosis in accordance with the invention. Macrophages recognize TSP-1 on
apoptotic cells via the
CD36 molecule, which is present on the surface of macrophages and may also be
present on apoptotic
cells. While not wishing to be bound by any theory, it is possible that
CD36/TSP 1 complex on the surface
of an apoptotic cell may form a ligand bridging the cell to a complex
consisting of alpha(v)beta
3/CD36/TSP1 on macrophages. It is possible that binding of TSP-1 to CD36 is
mediated by interaction of
the TSR-1 domain of TSP-1 with a conserved domain called CLESH-1 in CD36. In
certain embodiments
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of the invention phagocytosis is enhanced by increasing the level or density
of TSP-I, CD36, or a TSP-
1/CD36 complex on the surface of a cell or molecule, e.g., by delivering the
TSP-I, CD36, or TSP-
1/CD36 complex to the cell. In certain embodiments of the invention a TSP-
1/CLESH domain complex is
delivered to the cell.
[0077] Alternatively or additionally, the phagocytic marker may comprise a
molecule (e.g., MFG-E8, b2-
glycoprotein, etc.) that serves as a bridging agent between macrophages and
their targets, or a portion of
such a molecule. Such markers may, for example, facilitate recognition of
phosphatidyl serine by
macrophages or be independently recognized. Other markers that are also known
to enhance phagocytosis
include protein S, the growth arrest specific gene product GAS-6, and various
complement components
including, but not limited to, factor B, Clq, and C3. As mentioned above, MFG-
E8 is a secreted
glycoprotein, which is produced by stimulated macrophages and binds
specifically to apoptotic cells by
recognizing aminophospholipids such as phosphatidylserine (PS). MFG-E8, when
engaged by
phospholipids, binds to cells via its RGD (arginine-glycine-aspartate) motif
and binds particularly
strongly to cells expressing alpha(v)beta(3) integrin, such as macrophages. At
least two splice variants of
MFG-E8 are known, of which the L variant is believed to be active for
stimulating phagocytosis. In
certain embodiments of the invention the phagocytic marker comprises the L
splice variant of MFG-E8
(MFG-E8-L). In certain embodiments of the invention the phagocytic marker
comprises an N-terminal
domain of MFG-E8.
[0078] Annexin I is another phagocytic marker that may be used according to
the present invention.
Briefly, the 37 kDa protein annexin I (Anx-1; lipocortin 1) is a
glucocorticoid-regulated protein that has
been implicated in the regulation of phagocytosis, cell signaling and
proliferation, and is postulated to be a
mediator of glucocorticoid action in inflammation and in the control of
anterior pituitary hormone release.
Annexin I expression is elevated in apoptotic cells and appears to play a role
in bridging
phosphatidylserine on apoptotic cells to phagocytes and to enhancing
recognition of apoptotic cells by
phagocytes such as macrophages. While not wishing to be bound by any theory,
it is possible that the
phosphatidylserine receptor on macrophages recognizes either annexin I or a
complex containing annexin
I and PS, or that annexin I facilitates recognition by aggregating PS into
clusters. Additionally, other DC
targeting studies use conjugated targeting ligands such as anti-Dec-205 and
anti-CD11c to increase DC
specificity.
[0079] In some embodiments, the apoptotic signaling molecule may be conjugated
to the antigen-specific
peptide. In some instances, the apoptotic signaling molecule and antigen-
specific peptide are conjugated
by the creation of a fusion protein. As used herein, a "fusion protein" refers
to a protein formed by the
fusion of at least one antigen-specific peptide (or a fragment or a variant
thereof) to at least one molecule
of an apoptotic signaling molecule (or a fragment or a variant thereof). For
the creation of fusion proteins,
the terms "fusion protein," "fusion peptide," "fusion polypeptide," and
"chimeric peptide" are used
interchangably. Suitable fragments of the antigen-specific peptide include any
fragment of the full-length
peptide that retains the function of generating the desired antigen-specific
tolerance function of the present
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invention. Suitable fragments of the apoptotic signaling molecules include any
fragment of the full-length
peptide that retains the function of generating an apoptotic signal. The
present application is also directed
to proteins containing polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%
or 99% identical to the
reference polypeptide sequence (e.g., the antigen-specific peptide or
apoptotic signaling molecule or the
fusion protein thereof) set forth herein, or fragments thereof. Variant"
refers to a polynucleotide or
nucleic acid differing from a reference nucleic acid or polypeptide, but
retaining essential properties
thereof. Generally, variants are overall closely similar, and, in many
regions, identical to the reference
nucleic acid or polypeptide. As used herein, "variant", refers to an antigen-
specific peptide, apoptotic
signaling molecule or fusion protein thereof differing in sequence from an
antigen-specific peptide,
apoptotic signaling molecule or fusion protein thereof of the invention,
respectively, but retaining at least
one functional and/or therapeutic property thereof (e.g., trigger tolerance in
an immune system or produce
an apoptotic signal). The present invention is also directed to proteins which
comprise, or alternatively
consist of, an amino acid sequence which is at least 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99% or
100%, identical to, for example, the amino acid sequence of an antigen-
specific peptide, apoptotic
signaling molecule or fusion protein thereof of the invention.
[0080] The fusion protein may be created by various means. One means is by
genetic fusion (i.e. the
fusion protein is generated by translation of a nucleic acid sequence in which
a polynucleotide encoding
all or a portion or a variant of an antigen-specific peptide in joined in
frame to a polynucleotide encoding
all or a portion or a variant of an apoptotic signaling molecule. The two
proteins may be fused either
directly or via an amino acid linker. The polypeptides forming the fusion
protein are typically linked C-
terminus to N-terminus, although they can also be linked C-terminus to C-
terminus, N-terminus to N-
terminus, or N-terminus to C-terminus. The polypeptides of the fusion protein
can be in any order. This
term also refers to conservatively modified variants, polymorphic variants,
alleles, mutants, subsequences,
and interspecies homologs of the antigens that make up the fusion protein. The
fusion protein may also be
created by chemical conjugation. Protocols for generation of fusion
polypeptides are well known in the
art, and include various recombinant means and DNA synthesizers.
Alternatively, the apoptotic signaling
molecule and antigen-specific peptide fusion protein can also be easily
created using PCR amplification
and anchor primers that give rise to complementary overhangs between two
consecutive gene fragments
that can subsequently be annealed and reamplified to generate a chimeric gene
sequence. For example, an
apoptotic signaling molecule can be fused in-frame with an antigen-specific
peptide. In the present
invention, either the apoptotic signaling molecule or antigen-specific peptide
may be the N-terminal
portion of the fusion protein.
[00811 Fusion proteins may generally be prepared using standard techniques,
including chemical
conjugation. Preferably, a fusion protein is expressed as a recombinant
protein, allowing the production of
increased levels, relative to a non-fused protein, in an expression system.
Briefly, DNA sequences
encoding the polypeptide components may be assembled separately, and ligated
into an appropriate
expression vector. The 3' end of the DNA sequence encoding one polypeptide
component is ligated, with
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or without a peptide linker, to the 5' end of a DNA sequence encoding the
second polypeptide component
so that the reading frames of the sequences are in phase. This permits
translation into a single fusion
protein that retains the biological activity of both component polypeptides.
[0082] A peptide linker sequence may be employed to separate the first and
second polypeptide
components by a distance sufficient to ensure that each polypeptide folds into
its secondary and tertiary
structures. Such a peptide linker sequence is incorporated into the fusion
protein using standard
techniques well known in the art. Suitable peptide linker sequences may be
chosen based on the following
factors: (1) their ability to adopt a flexible extended conformation; (2)
their inability to adopt a secondary
structure that could interact with functional epitopes on the first and second
polypeptides; and (3) the lack
of hydrophobic or charged residues that might react with the polypeptide
functional epitopes. Preferred
peptide linker sequences contain Gly, Asn and Ser residues. Other near neutral
amino acids, such as Thr
and Ala may also be used in the linker sequence. Amino acid sequences which
may be usefully employed
as linkers include those disclosed in Maratea et. al., Gene 40:39-46 (1985);
Murphy et al., Proc. Natl.
Acad. Sci. USA 83:8258-8262 (1986); U.S. Pat. No. 4,935,233 and U.S. Pat. No.
4,751,180. The linker
sequence may generally be from 1 to about 50 amino acids in length. Linker
sequences are not required
when the first and second polypeptides have non-essential N-terminal amino
acid regions that can be used
to separate the functional domains and prevent steric interference.
[0083] The ligated DNA sequences are operably linked to suitable
transcriptional or translational
regulatory elements. The regulatory elements responsible for expression of DNA
are located only 5' to the
DNA sequence encoding the first polypeptides. Similarly, stop codons required
to end translation and
transcription termination signals are only present 3' to the DNA sequence
encoding the second
polypeptide.
Antigenic peptides and proteins
[0084] The practitioner has a number of choices for antigens used in the
combinations of this invention.
The inducing antigen present in the combination contributes to the specificity
of the tolerogenic response
that is induced. It may or may not be the same as the target antigen, which is
the antigen present or to be
placed in the subject being treated which is a target for the unwanted
immunological response, and for
which tolerance is desired.
[0085] An inducing antigen of this invention may be a polypeptide,
polynucleotide, carbohydrate,
glycolipid, or other molecule isolated from a biological source, or it may be
a chemically synthesized
small molecule, polymer, or derivative of a biological material, providing it
has the ability to induce
tolerance according to this description when combined with the mucosal binding
component.
[0086] In certain embodiments of this invention, the inducing antigen is a
single isolated or
recombinantly produced molecule. For treating conditions where the target
antigen is disseminated to
various locations in the host, it is generally necessary that the inducing
antigen be identical to or
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immunologically related to the target antigen. Examples of such antigens are
most polynucleotide
antigens, and some carbohydrate antigens (such as blood group antigens).
[0087] Where the target antigen is preferentially expressed on a particular
organ, cell, or tissue type, the
practitioner again has the option of using an inducing antigen which is
identical with or immunologically
related to the target antigen. However, there is also the additional option of
using an antigen which is a
bystander for the target. This is an antigen which may not be immunologically
related to the target
antigen, but is preferentially expressed in a tissue where the target antigen
is expressed. A working theory
as to the effectiveness of bystander suppression is that suppression is an
active cell-mediated process that
down-regulates the effector arm of the immune response at the target cells.
The suppressor cells are
specifically stimulated by the inducer antigen at the mucosal surface, and
home to a tissue site where the
bystander antigen is preferentially expressed. Through an interactive or
cytokine-mediated mechanism,
the localized suppressor cells then down-regulate effector cells (or inducers
of effector cells) in the
neighborhood, regardless of what they are reactive against. If the effector
cells are specific for a target
different from the inducing antigen, then the result is a bystander effect.
For further elaboration of the
bystander reaction and a list of tolerogenic peptides having this effect, the
reader is referred to
International Patent Publication WO 93/16724. An implication of bystander
theory is that one of ordinary
skill need not identify or isolate a particular target antigen against which
tolerance is desired in order to
practice the present invention. The practitioner need only be able to obtain
at least one molecule
preferentially expressed at the target site for use as an inducing antigen.
[0088] In certain embodiments of this invention, the inducing antigen is not
in the same form as
expressed in the individual being treated, but is a fragment or derivative
thereof. Inducing antigens of this
invention include peptides based on a molecule of the appropriate specificity
but adapted by
fragmentation, residue substitution, labeling, conjugation, and/or fusion with
peptides having other
functional properties. The adaptation may be performed for any desirable
purposes, including but not
limited to the elimination of any undesirable property, such as toxicity or
immunogenicity; or to enhance
any desirable property, such as mucosal binding, mucosal penetration, or
stimulation of the tolerogenic
arm of the immune response. Terms such as insulin peptide, collagen peptide,
and myelin basic protein
peptide, as used herein, refer not only to the intact subunit, but also to
allotypic and synthetic variants,
fragments, fusion peptides, conjugates, and other derivatives that contain a
region that is homologous
(preferably 70% identical, more preferably 80% identical and even more
preferably 90% identical at the.
amino acid level) to at least 10 and preferably 20 consecutive amino acids of
the respective molecule for
which it is an analog, wherein the homologous region of the derivative shares
with the respective parent
molecule an ability to induce tolerance to the target antigen.
[0089] It is recognized that tolerogenic regions of an inducing antigen are
often different from
immunodominant epitopes for the stimulation of an antibody response.
Tolerogenic regions are generally
regions that can be presented in particular cellular interactions involving T
cells. Tolerogenic regions may
be present and capable of inducing tolerance upon presentation of the intact
antigen. Some antigens
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contain cryptic tolerogenic regions, in that the processing and presentation
of the native antigen does not
normally trigger tolerance. An elaboration of cryptic antigens and their
identification is found in
International Patent Publication WO 94/27634.
[00901 In certain embodiments of this invention, two, three, or a higher
plurality of inducing antigens is
used. It may be desirable to implement these embodiments when there are a
plurality of target antigens, or
to provide a plurality of bystanders for the target. For example, both insulin
and glucagon can be mixed
with a mucosal binding component in the treatment of diabetes. It may also be
desirable to provide a
cocktail of antigens to cover several possible alternative targets. For
example, a cocktail of
histocompatibility antigen fragments could be used to tolerize a subject in
anticipation of future
transplantation with an allograft of unknown phenotype. Allovariant regions of
human leukocyte antigens
are known in the art: e.g., Immunogenetics 29:231, 1989. In another example, a
mixture of allergens may
serve as inducing antigen for the treatment of atopy.
[0091] Inducing antigens can be prepared by a number of techniques known in
the art, depending on the
nature of the molecule. Polynucleotide, polypeptide, and carbohydrate antigens
can be isolated from cells
of the species to be treated in which they are enriched. Short peptides are
conveniently prepared by amino
acid synthesis. Longer proteins of known sequence can be prepared by
synthesizing an encoding sequence
or PCR-amplifying an encoding sequence from a natural source or vector, and
then expressing the
encoding sequence in a suitable bacterial or eukaryotic host cell.
10092] In certain embodiments of this invention, the combination comprises a
complex mixture of
antigens obtained from a cell or tissue, one or more of which plays the role
of inducing antigen. The
antigens may be in the form of whole cells, either intact or treated with a
fixative such as formaldehyde,
glutaraldehyde, or alcohol. The antigens may be in the form of a cell lysate,
created by detergent
solubilization or mechanical rupture of cells or tissue, followed by
clarification. The antigens may also be
obtained by subcellular fractionation, particularly an enrichment of plasma
membrane by techniques such
as differential centrifugation, optionally followed by detergent
solubilization and dialysis. Other
separation techniques are also suitable, such as affinity or ion exchange
chromatography of solubilized
membrane proteins.
[0093] In one embodiment, the antigenic peptide or protein is an autoantigen,
an alloantigen or a
transplantation antigen. In yet another particular embodiment, the autoantigen
is selected from the group
consisting of myelin basic protein, collagen or fragments thereof, DNA,
nuclear and nucleolar proteins,
mitochondrial proteins and pancreatic 0-cell proteins.
[0094] The invention provides for the induction of tolerance to an autoantigen
for the treatment of
autoimmune diseases by administering the antigen for which tolerance is
desired. For example,
autoantibodies directed against the myelin basic protein (MBP) are observed in
patients with multiple
sclerosis, and, accordingly, MBP antigenic peptides or proteins may be used in
the invention to be
delivered using the compositions of the present invention to treat and prevent
multiple sclerosis.
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[0095] By way of another non-limiting example, an individual who is a
candidate for a transplant from a
non-identical twin may suffer from rejection of the engrafted cells, tissues
or organs, as the engrafted
antigens are foreign to the recipient. Prior tolerance of the recipient
individual to the intended graft
abrogates or reduces later rejection. Reduction or elimination of chronic anti-
rejection therapies may be
achieved by the practice of the present invention. In another example, many
autoimmune diseases are
characterized by a cellular immune response to an endogenous or self antigen.
Tolerance of the immune
system to the endogenous antigen is desirable to control the disease.
[0096] In a further example, sensitization of an individual to an industrial
pollutant or chemical, such as
may be encountered on-the-job, presents a hazard of an immune response. Prior
tolerance of the
individual's immune system to the chemical, in particular in the form of the
chemical reacted with the
individual's endogenous proteins, may be desirable to prevent the later
occupational development of an
immune response.
[0097] Allergens are other antigens for which tolerance of the immune response
thereto is also desirable.
[0098] Notably, even in diseases where the pathogenic autoantigen is unknown,
bystander suppression
may be induced using antigens present in the anatomical vicinity. For example,
autoantibodies to collagen
are observed in rheumatoid arthritis and, accordingly, a collagen-encoding
gene may be utilized as the
antigen-expressing gene module in order to treat rheumatoid arthritis (see
e.g. Choy (2000) Curr Opin
Investig Drugs 1: 58-62). Furthermore, tolerance to beta cell autoantigens may
be utilized to prevent
development of type 1 diabetes (see e.g. Bach and Chatenoud (2001) Ann Rev
Immunol 19: 131-161).
[0099] As another example, auto-antibodies directed against myelin
oligodendrocyte glycoprotein
(MOG) is observed in autoimmune encephalomyelitis and in many other CNS
diseases as well as multiple
sclerosis (see e.g. Iglesias et al. (2001) Glia 36: 22-34). Accordingly, use
of MOG antigen expressing
constructs in the invention allows for treatment of multiple sclerosis as well
as related autoimmune
disorders of the central nervous system.
[00100] Still other examples of candidate autoantigens for use in treating
autoimmune disease include:
pancreatic beta-cell antigens, insulin and GAD to treat insulin-dependent
diabetes mellitus; collagen type
11, human cartilage gp 39 (HCgp39) and gp130-RAPS for use in treating
rheumatoid arthritis; myelin
basic protein (MBP), proteolipid protein (PLP) and myelin oligodendrocyte
glycoprotein (MOG, see
above) to treat multiple sclerosis; fibrillarin, and small nucleolar protein
(snoRNP) to treat scleroderma;
thyroid stimulating factor receptor (TSH-R) for use in treating Graves'
disease; nuclear antigens, histories,
glycoprotein gp70 and ribosomal proteins for use in treating systemic lupus
erythematosus; pyruvate
dehydrogenase dehydrolipoamide acetyltransferase (PCD-E2) for use in treating
primary billiary cirrhosis;
hair follicle antigens for use in treating alopecia areata; and human
tropomyosin isoform 5 (hTM5) for use
in treating ulcerative colitis.
Evaluating tolerance
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[00101] Combinations can be tested for their ability to promote tolerance by
conducting experiments
with isolated cells or in animal models.
[00102] A proxy for tolerogenic activity is the ability of an intact antigen
or fragment to stimulate the
production of an appropriate cytokine at the target site. The immunoregulatory
cytokine released by T
suppressor cells at the target site is thought to be TGF-(3 (Miller et al.,
Proc. Natl. Acad. Sci. USA 89:421,
1992). Other factors that may be produced during tolerance are the cytokines
1L4 and IL-10, and the
mediator PGE. In contrast, lymphocytes in tissues undergoing active immune
destruction secrete
cytokines such as IL-l, IL-2, IL-6, and y-IFN. Hence, the efficacy of a
candidate inducing antigen can be
evaluated by measuring its ability to stimulate the appropriate type of
cytokines.
[00103] With this in mind, a rapid screening test for tolerogenic epitopes of
the inducing antigen,
effective mucosal binding components, effective combinations, or effective
modes and schedules of
mucosal administration can be conducted using syngeneic animals as donors for
in vitro cell assays.
Animals are treated at a mucosal surface with the test composition, and at
some time are challenged with
parenteral administration of the target antigen in complete Freund's adjuvant.
Spleen cells are isolated, and
cultured in vitro in the presence of the target antigen at a concentration of
about 50 tg/mL. Target antigen
can be substituted with candidate proteins or sub-fragments to map the
location of tolerogenic epitopes.
Cytokine secretion into the medium can be quantitated by standard immunoassay.
[00104] The ability of the cells to suppress the activity of other cells can
be determined using cells
isolated from an animal immunized with the target antigen, or by creating a
cell line responsive to the
target antigen (Ben-Nun et al., Eur. J. Immunol. 11:195, 1981). In one
variation of this experiment, the
suppressor cell population is mildly irradiated (about 1000 to 1250 rads) to
prevent proliferation, the
suppressors are co-cultured with the responder cells, and then tritiated
thymidine incorporation (or MTT)
is used to quantitate the proliferative activity of the responders. In another
variation, the suppressor cell
population and the responder cell population are cultured in the upper and
lower levels of a dual chamber
transwell culture system (Costar, Cambridge Mass.), which permits the
populations to coincubate within 1
mm of each other, separated by a polycarbonate membrane (WO 93/16724). In this
approach, irradiation
of the suppressor cell population is unnecessary, since the proliferative
activity of the responders can be
measured separately.
[00105] In embodiments of the invention where the target antigen is already
present in the individual,
there is no need to isolate the antigen or precombine it with the mucosal
binding component. For example,
the antigen may be expressed in the individual in a certain fashion as a
result of a pathological condition
(such as inflammatory bowel disease or Celiac disease) or through digestion of
a food allergen. Testing is
performed by giving the mucosal binding component in one or more doses or
formulations, and
determining its ability to promote tolerization against the antigen in situ.
[00106] The effectiveness of compositions and modes of administration for
treatment of specific
disease can also be elaborated in a corresponding animal disease model. The
ability of the treatment to
diminish or delay the symptomatology of the disease is monitored at the level
of circulating biochemical
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and immunological hallmarks of the disease, immunohistology of the affected
tissue, and gross clinical
features as appropriate for the model being employed. Non-limiting examples of
animal models that can
be used for testing are included in the following section.
[00107] The invention contemplates modulation of tolerance by modulating TH1
response, TH2
response, TH17 response, or a combination of these responses. Modulating TH1
response encompasses
changing expression of, e.g., interferon-gamma. Modulating TH2 response
encompasses changing
expression of, e.g., any combination of IL-4, IL-5, IL-10, and IL-13.
Typically an increase (decrease) in
TH2 response will comprise an increase (decrease) in expression of at least
one of IL-4, IL-5, IL-10, or
IL-13; more typically an increase (decrease) in TH2 response will comprise an
increase in expression of at
least two of IL-4, IL-5, IL-10, or IL-13, most typically an increase
(decrease) in TH2 response will
comprise an increase in at least three of IL-4, IL-5, IL- 10, or IL-13, while
ideally an increase (decrease) in
TH2 response will comprise an increase (decrease) in expression of all of IL-
4, IL-5, IL-10, and IL- 13.
Modulating TH 17 encompasses changing expression of, e.g., TGF-beta, IL-6, IL-
21 and IL23, and effects
levels of IL-17, IL-21 and IL-22.
[00108] In the study of the present invention, despite accelerated disease
onset, overall disease
incidence and severity was reduced over time in CD200KO mice, where reduction
in disease symptoms
correlated with elevated numbers of regulatory T cells and the presence of
high IL-10 secreting splenic
myeloid cells later in the disease process. The CD200KO enhanced tolerance to
retinal antigen. This result
of the CD200KO may be related to the altered phenotype of APC in the
respiratory tract compared to wild
type and an enhanced Th2 switch in tolerised CD200KO mice. Tolerance induction
in the CD200KO
mouse was efficient, with up 50% of eyes still protected from disease 28 days
post-immunisation (see,
e.g., Murphy and Reiner (2002) Nat. Rev. Immunol. 2:933-944; Sun-Payer, et at.
(1998) J. Immunol.
160:1212-1218; Thornton and Shevach (2000) J. Immunol. 164:183-190; Roncarolo,
et al. (2001)
Immunol. Rev. 182:68-79; Peiser and Gordon (2001) Microbes Infect. 3:149-159;
Gordon (2003) Nat.
Rev. Immuno l . 3:23-35).
[00109] In the studies of the present invention, there was a clear increase in
CD1 1b-ILIOh'gh cells in
the spleens of both sham tolerised and tolerised CD200KO mice at day 28. These
cells were distinct from
larger populations of CD11b'1L101o" present in all experimental groups from
day 21. The high level of IL-
detected was endogenous as cells were analysed directly ex vivo without any
additional activating
stimulus or artificial sequestering of cytokine by brefeldin A or other Golgi
inhibitors. Further analysis of
these cells indicated that they were CD 11 c-/low, CD45RB`ntermediate and B220-
and had plasmacytoid DC
morphology. Tolerogenic plasmacytoid DC with similar phenotype but CD45RBh'gh
can be generated by
in vitro culture with IL-I 0, can be isolated from the spleens of normal C57B
1/6 mice and are elevated in
IL10 transgenic mice. The cells take 3 weeks to differentiate in vitro, and in
the studies of the present
invention appear in CD200KO spleens 3-4 weeks after disease onset suggesting
that prolonged
stimulation and/or several rounds of cell division are involved. Bone marrow
derived plasmacytoid cells
were also tolerogenic and capable of generating antigen specific IL-10
secreting Tregs, in vivo.
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Significant numbers of CD3 +CD4+IL- 10+ cells were not found in this study,
but a trend towards increased
numbers of CD3+CD4+CD25+ in CD200KO mice was found and this was significant in
tolerised groups at
all time points. Tregs can have an immunosuppressive effect, e.g., by
inhibiting expression of IL-2 or IL-
10. Induction of IL-10 and suppression of IL-2 in all groups at day 28 of the
study of the present invention
is consistent with induction of regulatory T cells during the disease process,
and findings linking nasal
administration of antigen with induction of Trl (see, e.g., Shevach (2002)
Nat. Rev. Immunol 2:389400;
McGuirk and Mills (2002) Trends Immunol. 23:450455; Herrath and Harrison
(2003) Nat. Rev. Immunol.
3:223-232; Bluestone and Abbas (2003) Nat. Rev. Immunol. 3:253-257; Thornton
and Shevach (1998) J.
Exp. Med. 188:287-296; Jonuleit, et al. (2000) J. Exp. Med. 192:1213-1222;
Wakkach, et al. (2003)
Immunity 18:605-617).
[001101 Tolerance to autoantigens and autoimmune disease is achieved by a
variety of mechanisms
including negative selection of self-reactive T cells in the thymus and
mechanisms of peripheral tolerance
for those autoreactive T cells that escape thymic deletion and are found in
the periphery. Examples of
mechanisms that provide peripheral T cell tolerance include "ignorance" of
self antigens, anergy or
unresponsiveness to autoantigen, cytokine immune deviation, and activation-
induced cell death of self-
reactive T cells. In addition, regulatory T cells have been shown to be
involved in mediating peripheral
tolerance. See, for example, Walker et al. (2002) Nat. Rev. Immunol. 2:11-19;
Shevach et al. (2001)
Immunol. Rev. 182:58-67. In some situations, peripheral tolerance to an
autoantigen is lost (or broken)
and an autoimmune response ensues. For example, in an animal model for EAE,
activation of antigen
presenting cells (APCs) through TLR innate immune receptors was shown to break
self-tolerance and
result in the induction of EAE (Waldner et al. (2004) J. Clin. Invest. 113:990-
997).
[001111 Accordingly, in some embodiments, the invention provides methods for
increasing antigen
presentation while suppressing or reducing TLR7/8, TLR9, and/or TLR 7/8/9
dependent cell stimulation.
As described herein, administration of particular NISCs results in antigen
presentation by DCs or APCs
while suppressing the TLR 7/8, TLR9, and/ot TLR7/8/9 dependent cell responses
associated with
immunostimulatory polynucleotides. Such suppression may include decreased
levels of one or more TLR-
associated cytokines. IRPs appropriate for use in suppressing TLR9 dependent
cell stimulation are those
IRP that inhibit or suppress cell responses associated with TLR9.
Methods of use
[001121 The invention provides methods of regulating an immune response in an
individual,
preferably a mammal, more preferably a human, comprising administering to the
individual an antigen-
carrier complex as described herein. Methods of immunoregulation provided by
the invention include
those that suppress and/or inhibit an innate immune response, including, but
not limited to, an immune
response stimulated by immunostimulatory polypeptides, such as myelin basic
protein.
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[00113] The antigen-carrier complex is administered in an amount sufficient to
regulate an immune
response. As described herein, regulation of an immune response may be humoral
and/or cellular, and is
measured using standard techniques in the art and as described herein.
[00114] In certain embodiments, the individual suffers from a disorder
associated with unwanted
immune activation, such as allergic disease or condition, allergy and asthma.
An individual having an
allergic disease or asthma is an individual with a recognizable symptom of an
existing allergic disease or
asthma.
[00115] In certain embodiments, the individual suffers from a disorder
associated with unwanted
immune activation, such as autoimmune disease and inflammatory disease. An
individual having an
autoimmune disease or inflammatory disease is an individual with a
recognizable symptom of an existing
autoimmune disease or inflammatory disease.
[00116] Autoimmune diseases can be divided in two broad categories: organ-
specific and systemic.
Autoimmune diseases include, without limitation, rheumatoid arthritis (RA),
systemic lupus
erythematosus (SLE), type I diabetes mellitus, type II diabetes mellitus,
multiple sclerosis (MS), immune-
mediated infertility such as premature ovarian failure, scleroderma, Sjogren's
disease, vitiligo, alopecia
(baldness), polyglandular failure, Grave's disease, hypothyroidism,
polymyositis, pemphigus vulgaris,
pemphigus foliaceus, inflammatory bowel disease including Crohn's disease and
ulcerative colitis,
autoimmune hepatitis including that associated with hepatitis B virus (HBV)
and hepatitis C virus (HCV),
hypopituitarism, graft-versus-host disease (GvHD), myocarditis, Addison's
disease, autoimmune skin
diseases, uveitis, pernicious anemia, and hypoparathyroidism.
[00117] Autoimmune diseases may also include, without limitation, Hashimoto's
thyroiditis, Type I
and Type II autoinimune polyglandular syndromes, paraneoplastic pemphigus,
bullus pemphigoid,
dermatitis herpetiformis, linear IgA disease, epidermolysis bullosa acquisita,
erythema nodosa,
pemphigoid gestations, cicatricial pemphigoid, mixed essential
cryoglobulinemia, chronic bullous disease
of childhood, hemolytic anemia, thrombocytopenic purpura, Goodpasture's
syndrome, autoimmune
neutropenia, myasthenia gravis, Eaton-Lambert myasthenic syndrome, stiff-man
syndrome, acute
disseminated encephalomyelitis, Guillain-Barre syndrome, chronic inflammatory
demyelinating
polyradiculoneuropathy, multifocal motor neuropathy with conduction block,
chronic neuropathy with
monoclonal gammopathy, opsonoclonus-myoclonus syndrome, cerebellar
degeneration,
encephalomyelitis, retinopathy, primary biliary sclerosis, sclerosing
cholangitis, gluten-sensitive
enteropathy, ankylosing spondylitis, reactive arthritides,
polymyositis/dermatomyositis, mixed connective
tissue disease, Bechet's syndrome, psoriasis, polyarteritis nodosa, allergic
anguitis and granulomatosis
(Churg-Strauss disease), polyangiitis overlap syndrome, hypersensitivity
vasculitis, Wegener's
granulomatosis, temporal arteritis, Takayasu's arteritis, Kawasaki's disease,
isolated vasculitis of the
central nervous system, thromboangiutis obliterans, sarcoidosis,
glomerulonephritis, and cryopathies.
These conditions are well known in the medical arts and are described, for
example, in Harrison's
Principles of Internal Medicine, 14th ed., Fauci A S et al., eds., New York:
McGraw-Hill, 1998.
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[001181 In some embodiments, the invention relates to uses of compositions of
this invention prior to
the onset of disease. In other embodiments, the invention relates to uses of
the compositions of this
invention to inhibit ongoing disease. In some embodiments, the invention
relates to ameliorating disease
in a subject. By ameliorating disease in a subject is meant to include
treating, preventing or suppressing
the disease in the subject.
[00119] In some embodiments, the invention relates to preventing the relapse
of disease. For
example, an unwanted immune response can occur at one region of a peptide
(such as an antigenic
determinant). Relapse of a disease associated with an unwanted immune response
can occur by having an
immune response attack at a different region of the peptide. T-cell responses
in some immune response
disorders, including MS and other Thl/17-mediated autoimmune diseases, can be
dynamic and evolve
during the course of relapsing-remitting and/or chronic-progressive disease.
The dynamic nature of the T-
cell repertoire has implications for treatment of certain diseases, since the
target may change as the
disease progresses. Previously, pre-existing knowledge of the pattern of
responses was necessary to
predict the progression of disease. The present invention provides
compositions that can prevent the
effect of dynamic changing disease, a function of "epitope spreading." A known
model for relapse is an
immune reaction to proteolipid protein (PLP) as a model for multiple sclerosis
(MS). Initial immune
response can occur by a response to PLP139-151 = Subsequent disease onset can
occur by a relapse immune
response to PLP17s-191= Compositions of the present invention have been shown
to prevent relapse of
disease using the PLP model.
[00120] Certain embodiments of this invention relate to priming of immune
tolerance in an individual
not previously tolerized by therapeutic intervention. These embodiments
generally involve a plurality of
administrations of a combination of antigen and mucosal binding component.
Typically, at least three
administrations, frequently at least four administrations, and sometimes at
least six administrations are
performed during priming in order to achieve a long-lasting result, although
the subject may show
manifestations of tolerance early in the course of treatment. Most often, each
dose is given as a bolus
administration, but sustained formulations capable of mucosal release are also
suitable. Where multiple
administrations are performed, the time between administrations is generally
between 1 day and 3 weeks,
and typically between about 3 days and 2 weeks. Generally, the same antigen
and mucosal binding
component are present at the same concentration, and the administration is
given to the same mucosal
surface, but variations of any of these variables during a course of treatment
may be accommodated.
[001211 Other embodiments of this invention relate to boosting or extending
the persistence of a
previously established immune tolerance. These embodiments generally involve
one administration or a
short course of treatment at a time when the established tolerance is
declining or at risk of declining.
Boosting is generally performed 1 month to 1 year, and typically 2 to 6 months
after priming or a previous
boost. This invention also includes embodiments that involve regular
maintenance of tolerance on a
schedule of administrations that occur semiweekly, weekly, biweekly, or on any
other regular schedule.
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[00122] Other embodiments of this invention relate to treatment of
pathological conditions relating to
an unwanted hypersensitivity. The hypersensitivity can be any one of types I,
II, III, and IV. Immediate
(type I) hypersensitivity is typically treated by using one or more offending
allergen or tolerogenic
fragments thereof as the inducing antigen. The frequency of administration
will typically correspond with
the timing of allergen exposure. Suitable animal models are known in the art
(for example, Gundel et al.,
Am. Rev. Respir. Dis. 146:369, 1992; Wada et al., J. Med. Chem. 39,2055,1996;
and WO 96/35418).
[00123] Other embodiments of this invention relate to transplantation. This
refers to the transfer of a
tissue sample or graft from a donor individual to a recipient individual, and
is frequently performed on
human recipients who need the tissue in order to restore a physiological
function provided by the tissue.
Tissues that are transplanted include (but are not limited to) whole organs
such as kidney, liver, heart,
lung; organ components such as skin grafts and the cornea of the eye; and cell
suspensions such as bone
marrow cells and cultures of cells selected and expanded from bone marrow or
circulating blood, and
whole blood transfusions.
[00124] A serious potential complication of any transplantation ensues from
antigenic differences
between the host recipient and the engrafted tissue. Depending on the nature
and degree of the difference,
there may be a risk of an immunological assault of the graft by the host, or
of the host by the graft, or
both, may occur. The extent of the risk is determined by following the
response pattern in a population of
similarly treated subjects with a similar phenotype, and correlating the
various possible contributing
factors according to well accepted clinical procedures. The immunological
assault may be the result of a
preexisting immunological response (such as preformed antibody), or one that
is initiated about the time
of transplantation (such as the generation of TH cells). Antibody, TH cells,
or Tc cells may be involved in
any combination with each other and with various effector molecules and cells.
[00125] It is an object of this invention to provide materials and procedures
that permit transplantation
to be conducted according to standard surgical procedures, but with decreased
risk of an adverse
immunological reaction to the recipient of the transplant. The procedures
involve tolerizing the recipient
to the tissues of the donor, or vice versa, or both. The tolerizing is
performed by administering a target
antigen expressed in the transplanted tissue, or a bystander antigen, in a
composition of the present
invention. The target antigen may be, for example, allogeneic cell extracts.
The graft may be a complex
structure of many different cell types, and any one or more of the cell types
transplanted into the
individual may pose a risk for which the procedures of this invention are
appropriate. For example,
endothelial cell antigens complicate renal transplants, and passenger
lymphocytes complicate hepatic
transplants.
[00126] Certain embodiments of the invention relate to decreasing the risk of
host versus graft disease,
leading to rejection of the tissue graft by the recipient. The treatment may
be performed to prevent or
reduce the effect of a hyperacute, acute, or chronic rejection response.
Treatment is preferentially initiated
sufficiently far in advance of the transplant so that tolerance will be in
place when the graft is installed;
but where this is not possible, treatment can be initiated simultaneously with
or following the transplant.
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Regardless of the time of initiation, treatment will generally continue at
regular intervals for at least the
first month following transplant. Follow-up doses may not be required if a
sufficient accommodation of
the graft occurs, but can be resumed if there is any evidence of rejection or
inflammation of the graft. Of
course, the tolerization procedures of this invention may be combined with
other forms of
immunosuppression to achieve an even lower level of risk.
[00127] Certain embodiments of this invention relate to decreasing the risk of
graft versus host
disease. In this series of embodiments, it is necessary to tolerize a living
donor against a target antigen of
the future graft recipient before the transplantation occurs. Once tolerance
is achieved, the cells or tissue
of the donor are harvested and the transplant is performed.
[00128] Animal models for the study of autoimmune disease are known in the
art. For example,
animal models which appear most similar to human autoimmune disease include
animal strains which
spontaneously develop a high incidence of the particular disease. Examples of
such models include, but
are not limited to, the nonobeses diabetic (NOD) mouse, which develops a
disease similar to type 1
diabetes, and lupus-like disease prone animals, such as New Zealand hybrid,
MRL-FasIpr and BXSB mice.
Animal models in which an autoimmune disease has been induced include, but are
not limited to,
experimental autoimmune encephalomyelitis (EAE), which is a model for multiple
sclerosis, collagen-
induced arthritis (CIA), which is a model for rheumatoid arthritis, and
experimental autoimmune uveitis
(EAU), which is a model for uveitis. Animal models for autoimmune disease have
also been created by
genetic manipulation and include, for example, IL-2/IL-10 knockout mice for
inflammatory bowel
disease, Fas or Fas ligand knockout for SLE, and IL-1 receptor antagonist
knockout for rheumatoid
arthritis.
Administration and Assessment of the Immune Response
[00129] The carrier can be administered in combination with other
pharmaceutical agents, as
described herein, and can be combined with a physiologically acceptable
carrier thereof (and as such the
invention includes these compositions). The carrier may be any of those
described herein.
[00130] Compositions of this invention can be prepared for administration to
an individual in need
thereof, particularly human subjects having an unwanted immune response. The
preparation of
compositions and their use is conducted in accordance with generally accepted
procedures for the
preparation of pharmaceutical compositions.
[00131] Procedures for preparing pharmaceutical compositions are described in
Remington's
Pharmaceutical Sciences, E. W. Martin ed., Mack Publishing Co., Pa. The
mucosal binding component
and the antigen (whether given separately or together) are optionally combined
with other active
components, carriers and excipients, and stabilizers. Additional active
components of interest are agents
that enhance the tolerogenic effect of the combination at the mucosal surface.
An example of an additional
active component is a cytokine, exemplified by IL-4. Although not required,
pharmaceutical compositions
can be supplied in unit dosage form suitable for administration of a precise
amount.
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[001321 The effective amounts and method of administration of the present
invention for modulation
of an immune response can vary based on the individual, what condition is to
be treated and other factors
evident to one skilled in the art. Factors to be considered include route of
administration and the number
of doses to be administered. Such factors are known in the art and it is well
within the skill of those in the
art to make such determinations without undue experimentation. A suitable
dosage range is one that
provides the desired regulation of immune response (e.g., suppression of IFN-
(x or other cytokine
production). Useful dosage ranges of the carrier, given in amounts of carrier
delivered, may be, for
example, from about any of the following: 0.5 to 10 mg/kg, 1 to 9 mg/kg, 2 to
8 mg/kg, 3 to 7 mg/kg, 4 to
6 mg/kg, 5 mg/kg, 1 to 10 mg/kg, 5 to 10 mg/kg. Alternatively, the dosage can
be administered based on
the number of particles. For example, useful dosages of the carrier, given in
amounts of carrier delivered,
may be, for example, from about any of the following: greater than 106, 107,
101, 109, or 1010 particles per
dose, or from 1x107 to 1x109 particles per dose, or from 1x108 to 1x109
particles per dose, or from 2x109
to 5x109 particles per dose. The absolute amount given to each patient depends
on pharmacological
properties such as bioavailability, clearance rate and route of
administration. Details of pharmaceutically
acceptable carriers, diluents and excipients and methods of preparing
pharmaceutical compositions and
formulations are provided in Remmingtons Pharmaceutical Sciences 18"1 Edition,
1990, Mack Publishing
Co., Easton, Pa., USA., which is hereby incorporated by reference in its
entirety.
[001331 The effective amount and method of administration of the particular
carrier formulation can
vary based on the individual patient, desired result and/or type of disorder,
the stage of the disease and
other factors evident to one skilled in the art. The route(s) of
administration useful in a particular
application are apparent to one of skill in the art. Routes of administration
include but are not limited to
topical, dermal, transdermal, transmucosal, epidermal, parenteral,
gastrointestinal, and nano-pharyngeal
and pulmonary, including transbronchial and transalveolar. A suitable dosage
range is one that provides
sufficient IRP-containing composition to attain a tissue concentration of
about 1-50 tM as measured by
blood levels. The absolute amount given to each patient depends on
pharmacological properties such as
bioavailability, clearance rate and route of administration.
[001341 The present invention provides carrier formulations suitable for
topical application including,
but not limited to, physiologically acceptable implants, ointments, creams,
rinses and gels. Exemplary
routes of dermal administration are those which are least invasive such as
transdermal transmission,
epidermal administration and subcutaneous injection.
[001351 Transdermal administration is accomplished by application of a cream,
rinse, gel, etc. capable
of allowing the carrier to penetrate the skin and enter the blood stream.
Compositions suitable for
transdermal administration include, but are not limited to, pharmaceutically
acceptable suspensions, oils,
creams and ointments applied directly to the skin or incorporated into a
protective carrier such as a
transdermal device (so-called "patch"). Examples of suitable creams, ointments
etc. can be found, for
instance, in the Physician's Desk Reference. Transdermal transmission may also
be accomplished by
iontophoresis, for example using commercially available patches which deliver
their product continuously
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through unbroken skin for periods of several days or more. Use of this method
allows for controlled
transmission of pharmaceutical compositions in relatively great
concentrations, permits infusion of
combination drugs and allows for contemporaneous use of an absorption
promoter.
[001361 Parenteral routes of administration include but are not limited to
electrical (iontophoresis) or
direct injection such as direct injection into a central venous line,
intravenous, intramuscular,
intraperitoneal, intradermal, or subcutaneous injection. Formulations of
carrier suitable for parenteral
administration are generally formulated in USP water or water for injection
and may further comprise pH
buffers, salts bulking agents, preservatives, and other pharmaceutically
acceptable excipients.
Immunoregulatory polynucleotide for parenteral injection may be formulated in
pharmaceutically
acceptable sterile isotonic solutions such as saline and phosphate buffered
saline for injection.
[001371 Gastrointestinal routes of administration include, but are not limited
to, ingestion and rectal
routes and can include the use of, for example, pharmaceutically acceptable
powders, pills or liquids for
ingestion and suppositories for rectal administration.
[001381 Naso-pharyngeal and pulmonary administration include are accomplished
by inhalation, and
include delivery routes such as intranasal, transbronchial and transalveolar
routes. The invention includes
formulations of carrier suitable for administration by inhalation including,
but not limited to, liquid
suspensions for forming aerosols as well as powder forms for dry powder
inhalation delivery systems.
Devices suitable for administration by inhalation of carrier formulations
include, but are not limited to,
atomizers, vaporizers, nebulizers, and dry powder inhalation delivery devices.
[001391 As is well known in the art, solutions or suspensions used for the
routes of administration
described herein can include any one or more of the following components: a
sterile diluent such as water
for injection, saline solution, fixed oils, polyethylene glycols, glycerine,
propylene glycol or other
synthetic solvents; antibacterial agents such as benzyl alcohol or methyl
parabens; antioxidants such as
ascorbic acid or sodium bisulfate; chelating agents such as
ethylenediaminetetraacetic acid; buffers such as
acetates, citrates or phosphates and agents for the adjustment of tonicity
such as sodium chloride or
dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or
sodium hydroxide. The
parenteral preparation can be enclosed in ampoules, disposable syringes or
multiple dose vials made of
glass or plastic.
[001401 As is well known in the art, pharmaceutical compositions suitable for
injectable use include
sterile aqueous solutions (where water soluble) or dispersions and sterile
powders for the extemporaneous
preparation of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers
include physiological saline, bacteriostatic water, Cremophor ELTM (BASF,
Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must be sterile
and can be fluid to the
extent that easy syringability exists. It can be stable under the conditions
of manufacture and storage and
must be preserved against the contaminating action of microorganisms such as
bacteria and fungi. The
carrier can be a solvent or dispersion medium containing, for example, water,
ethanol, polyol (for
example, glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), and suitable mixtures
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thereof. The proper fluidity can be maintained, for example, by the use of a
coating such as lecithin, by
the maintenance of the required particle size in the case of dispersion and by
the use of surfactants.
Prevention of the action of microorganisms can be achieved by various
antibacterial and antifungal
agents, for example, parabens, chlorobutanol, phenol, ascorbic acid,
thimerosal, and the like. Some
embodiments include isotonic agents, for example, sugars, polyalcohols such as
mannitol, sorbitol,
sodium chloride in the composition. Prolonged absorption of the injectable
compositions can be brought
about by including in the composition an agent which delays absorption, for
example, aluminum
monostearate and gelatin.
[00141] As is well known in the art, sterile injectable solutions can be
prepared by incorporating the
active compound(s) in the required amount in an appropriate solvent with one
or a combination of
ingredients enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are
prepared by incorporating the active compound into a sterile vehicle which
contains a basic dispersion
medium and the required other ingredients from those enumerated above. In the
case of sterile powders
for the preparation of sterile injectable solutions, the preferred methods of
preparation are vacuum drying
and freeze-drying which yields a powder of the active ingredient plus any
additional desired ingredient
from a previously sterile-filtered solution thereof.
[00142] Certain embodiments of this invention relate to kits and reagents in
which one or more
component is provided in a separate container, optionally with written
instructions, for assembly of a
pharmaceutical composition by the patient or the administering health
professional.
EXAMPLES
[00143] While preferred embodiments of the present invention have been shown
and described herein,
it will be obvious to those skilled in the art that such embodiments are
provided by way of example only.
Numerous variations, changes, and substitutions will now occur to those
skilled in the art without
departing from the invention. It should be understood that various
alternatives to the embodiments of the
invention described herein may be employed in practicing the invention. It is
intended that the following
claims define the scope of the invention and that methods and structures
within the scope of these claims
and their equivalents be covered thereby.
Example 1. Induction of tolerance to EAE relapse using PLP peptides conjugated
to polystyrene spheres.
[00144] Polystyrene microspheres were coupled either with encephalitogenic
epitopes or control
peptides to determine whether active EAE can be induced using artificial
carriers.
[00145] Methods for determination of inhibition of induced EAE followed
procedures as previously
described (Smith and Miller (2006) J. Autoimmun. 27:218-31; Turley and Miller
(2007) J Immunol.
178:2212-20). Briefly, SJL mice, 6-7 weeks old, were purchased from Harlan
Laboratories, Bethesda,
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MD. All mice were housed under specific pathogen-free conditions (SPF) in the
Northwestern University
Center for Comparative Medicine. Paralyzed animals were afforded easier access
to food and water.
[00146] Fluoresbrite YG Carboxylate microspheres were purchased from
Polysciences, Inc.
(Warrington, PA). Synthetic peptides PLP139-151 (HSLGKWLGHPDKF) and OVA323_339
(ISQAVHAAHAEINEAGR) were purchased from Genemed, San Francisco, CA. The
peptides were
coupled to the microspheres using ECDI to a specific number of active amino or
carboxyl sites on the
particles.
[00147] Polystyrene microsphere suspensions were coupled with peptides using
ethylene carbodiimide
(ECDI). Microspheres were washed 2X in PBS, resuspended at 3.2x 106/ml in PBS
with 1 mg/ml of each
peptide and 30.75 mg/ml ECDI (CalBiochem, La Jolla, CA), and incubated for 1 h
at 4 C with periodic
shaking. Peptide-coupled microspheres were then washed 3X in PBS, filtered
through a 70 m cell
strainer, and resuspended at 250x 106 microspheres/ml in PBS. Female mice 8-10
weeks old were
injected intravenously with 109 of the indicated Fluoresbrite Carboxylate YG
0.50 micron microspheres
coupled with PLP139_151 or OVA323_339 on day -7 relative to priming with
PLP139_151/Complete Freund's
Adjuvant (CFA) on day 0. Individual animals were observed every 1-3 days, and
clinical scores were
assessed on a scale of 0-4 as follows: 0 = no abnormality; 1 = limp tail or
hind limb weakness; 2 = limp
tail and hind limb weakness; 3 = partial hind limb paralysis; 4 = complete
hind limb paralysis. Data are
reported as the mean daily clinical score. Mice were observed for clinical
signs of EAE for an additional
40 days.
[00148] Mice were anesthetized and perfused with 30 ml PBS on the indicated
days post-
immunization. Spinal cords were removed by dissection, and 2- to 3-mm spinal
cord blocks were
immediately frozen in OCT (Miles Laboratories; Elkhart, IN) in liquid
nitrogen. The blocks were stored at
-80 C in plastic bags to prevent dehydration. Six micrometer thick cross-
sections from the lumbar region
(approximately L2-L3) were cut on a Reichert-Jung Cyocut CM1850 cryotome
(Leica, Deerfield, IL),
mounted on Superfrost Plus electrostatically charged slides (Fisher,
Pittsburgh, PA), air dried, and stored
at -80 C. Slides were stained using a Tyramide Signal Amplification (TSA)
Direct kit (NEN, Boston,
MA) according to manufacturer's instructions. Lumbar sections from each group
were thawed, air-dried,
fixed in 2% paraformaldehyde at room temperature, and rehydrated in lx PBS.
Nonspecific staining was
blocked using anti-CD 16/CD32, (FCIIUIIR, 2.4G2; BD PharMingen), and an
avidin/biotin blocking kit
(Vector Laboratories) in addition to the blocking reagent provided by the TSA
kit. Tissues were stained
with biotin-conjugated Abs anti-mouse CD4 (H129.19) (BD Biosciences, San Jose,
CA) and anti-mouse
F4/80 (BM8) (Caltag, Burlingame, CA). Sections were coverslipped with
Vectashield mounting medium
including DAPI (Vector Laboratories, Burlingame, CA). Slides were examined and
images were acquired
via epifluorescence using the SPOT RT camera (Diagnostic Instruments, Sterling
Heights, MI) and
Metamorph imaging software (Universal Imaging, Downingtown, PA). Eight non-
serial lumbar sections
from each sample per group were analyzed at 100x and 200x magnification.
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[00149] The results are shown in Figure 1. Fluoresbrite Carboxylate YG 0.50
micron polystyrene
Microspheres (Polysciences, Warrington, PA) coupled with PLP 139-151 via an
amino linkage, but not
with OVA323-339, were not only effective in providing significant protection
from induction of PLP139-
151/CFA-induced EAE but more importantly completely abrogated the initiation
of relapse of active EAE
in SJL mice, This may indicate that inclusion of an apoptotic signal on the
inert carrier may not be
necessary depending on the composition of the carrier beads.
Example 2. Formulation of PLG Microspheres
[00150] This example describes the formulation of poly(lactide-co-glycolide-)
(PLG) microspheres
suitable for encapsulating and delivering antigen-specific peptides. The
microspheres are prepared using a
double emulsion technique Q. H. Eldridge et al. Mol Immunol, 28:287-294, 1991;
S. Cohen et al. Pharm
Res, 8:713-720, 1991). RG502H is used as the polymer, and polyvinyl alcohol is
used as a stabilizer.
Encapsulation efficiency is found to increase with increasing PLG
concentration in the organic phase
(dichloromethane) (30-200 mg/ml), which also correlats with an increase in
median microsphere diameter
(about 1 to about 10 gm).
Example 3. Preparation of Liposomal Composition Containing myelin basic
protein
[00151] An optimal myelin basic protein (MBP)/liposome ratio is determined
empirically by methods
previously described (17). To prepare the MBP/lipid combination, the
components are first brought to
room temperature. The lipid [1,2-dilauroyl-sn-glycero-3-phosph- ocholine
(DLPC); Avanti Polar-Lipids,
Inc., Alabaster, Ala.] at a concentration of 120 mg/ml is dissolved in
tertiary-butanol (Fisher Scientific,
Houston, Tex.) then sonicated to obtain a clear solution. MBP at 40 mg/ml is
also dissolved in tertiary-
butanol and vortexed until all solids are dissolved. The two solutions are
then combined in equal amounts
(v:v) to achieve the desired ratio of MBP/liposome, mixing by vortexing,
frozen at -80 C. for 1-2 h, and
lyophilizing overnight to a dry powder prior to storing at -20 C. until
needed. Each treatment vial contains
75 mg MBP.
Example 4. Synthesis of Poly(Glu-Lys) Polymer
[00152] A polypeptide polymer suitable for use as a linking carrier is
poly(glutamic acid-lysine)
(poly(glutamyl-lysine) or poly(EK)). N-a-Fmoc glutamic acid y-benzyl ester
(Fmoc-Glu(OBzl)-OH) is
coupled to N-E-CBZ lysine t-butyl ester (H-Lys(Z)-tBu) (both reagents are
commercially available from
Calbiochem-Novabiochem, San Diego, Calif.) using diisopropylcarbodiimide and 1-
hydroxybenzotriazole. The resulting dipeptide, Fmoc-Glu(OBzl)-Lys(Z)-tBu, can
be deprotected using
piperidine followed by 95% trifluoroacetic acid to yield H-Glu(OBzl)-Lys(Z)-
OH. The dipeptide unit can
then be freely polymerized to form a mixture of varying chain lengths, by
carbodiimide or other
condensation. Alternatively, if a defined length is desired, deprotection of
the amino terminal with
piperidine to afford H-Glu(OBzl)-Lys(Z)-OtBu and deprotection of the carboxyl
terminal with 95%
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trifluoroacetic acid to afford Fmoc-Glu(OBzl)-Lys(Z)-OH enables condensation
of the two dipeptides
with carbodiimides to give Fmoc-Glu(OBzl)-Lys(Z)-Glu(OBzl)-Lys- (Z)-OtBu.
Repetition of this cycle
can give poly(Glu(OBzl)-Lys(Z)) of a defined length. For either the random
polymer or the defined-length
polymer, the benzyl protecting group on glutamic acid and the CBZ protecting
group on lysine can be
removed simultaneously using either H2/Pd or strong acids such as liquid HF or
trifluoromethanesulfonic
acid. This makes available both free amino and free carboxyl groups for use in
attaching antigen-specific
peptides and/or apoptotic signaling molecules. The free amino groups can be
reprotected with Boc, Bpoc
or Fmoc groups in order to prevent reaction during derivatization of the
carboxylate groups, by using
standard methods in the field of peptide chemistry.
Example 5. Antigen-specific peptide-conjugated Polymerized Liposomes
[00153] Antigen-specific peptides are conjugated to polymerized liposomes to
form antigen-specific
peptide-conjugated polymerized liposomes for use in induction of tolerance to
the antigen-specific
peptide.
[00154] Lipid components of. 60% pentacosadiynoic acid filler lipid, 29.5%
chelator lipid, 10% amine
terminated lipid and 0.5% biotinylated lipid are combined in the indicated
amounts and the solvents are
evaporated. Water is added to yield a solution that is 30 mM in acyl chains.
The lipid/water mixture is
sonicated for at least one hour. During sonication, the pH of the solution is
maintained between 7 and 8
with NaOH and the temperature maintained above the gel-liquid crystal phase
transition point by the heat
generated by sonication. The liposomes are transferred to a petri dish resting
on a bed of wet ice and
irradiated at 254 rim for at least one hour to polymerize. The polymerized
liposomes are collected after
passage through a 0.2 m filter. To form the antigen-specific peptide-
conjugated polymerized liposomes,
2.3 g avidin is combined with 14.9 g biotinylated antibody in phosphate
buffered saline in about 1:3
molar ratio and incubated at room temperature for 15 minutes. This solution is
combined with 150 L of
the above formed polymerized liposomes and incubated at 4 C. overnight to form
the antigen-specific
peptide-conjugated polymerized liposomes.
Example 6. Method of producing nanoparticles
[00155] Nanoparticles are synthesized by inverse emulsion polymerization. An
emulsion is created by
adding the PEG block copolymer emulsifier, PLURONIC F-127 (Sigma-Aldrich,
Buchs, Switzerland) and
the monomer propylene sulfide to ultrapure milliQ water under constant
stirring. The protected initiator
pentaerythritol tetrathioester is deprotected by mixing it with 0.20 mL of 0.5
M sodium methylate solution
under stirring for 10 min. Following deprotection, the initiator is then added
to the monomer emulsion and
min later 60 l of the base diaza[5.4.0]bicycycloundec-7-ene (DBU) is added to
the reaction and
allowed to stir continuously for 24 h under an inert atmosphere. The
nanoparticles are then exposed to air
in order to produce disulfide cross-linking.
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[00156] The nanoparticles are purified from remaining monomers, base, or free
PLURONIC by 2 days
of repeated dialysis with a 12-14 kDa MWCO membrane (Spectrum Laboratories,
Rancho Dominguez,
Calif.) against ultrapure milliQ water. The nanoparticle size distributions
are determined by the use of a
dynamic light scattering instrument (Malvern, Worcestershire, United Kingdom).
Example 7. Myelin Basic Protein Conjugation to Nanoparticles
[00157] Antigen conjugation to nanoparticles can be accomplished by
functionalizing Pluronic (a
block co-polymer of PEG and PPG) surface with proteins or peptides. A
conjugation scheme is presented
in this example for the protein antigen myelin basic protein (MBP) using a
free cysteine residue for
chemical conjugation. Other functionalities can be used in related schemes,
such as amines at the N-
terminus or on lysine residues. Antigen may also be adsorbed to nanoparticle
surfaces.
[00158] For conjugation of MBP to nanoparticles, Pluronic divinylsulfone is
synthesized to which
MPB is coupled via a free thiol group on MPB in a Michael addition reaction.
Synthetic details for both
steps are given below.
[00159] Pluronic F127 (Sigma), divinylsulfone (Fluka), sodium hydride
(Aldrich), toluene (VWR),
acetic acid (Fluka), diethylether (Fisher), dichloromethane (Fisher) and
Celite (Macherey Nagel) are used
as received. The reaction is carried out under argon (Messer). 1H NMR is
measured in deuterated
chloroform (Armar) and chemical shifts are given in ppm relative to internal
standard tetramethylsilane
(Armar) signal at 0.0 ppm.
[00160] A solution of Pluronic F-127 in toluene is dried by azeotropic
distillation using a Dean-Stark
trap. The solution is cooled in an ice bath sodium hydride is added. The
reaction mixture is stirred for 15
min and divinyl sulfone (Sigma-Aldrich) is added quickly. After stirring in
the dark for 5 days at room
temperature the reaction is quenched by adding acetic acid. After filtering
over celite and concentrating
the filtrate under reduced pressure to a small volume the product is
precipitated in 1 liter of ice-cold
diethylether. The solid is filtered off, dissolved in a minimum amount of
dichloromethane and precipitated
in ice-cold diethylether four times in total. The polymer is dried under
vacuum and stored under argon at -
20 C.
Example 8. Flow Cytometry & Analysis and In Vitro Nanoparticle
Internalization: Uptake by APCs,
including DCS
[00161] Flow cytometry analysis is performed to quantify the fraction of APCs
and DCs in lymph
nodes that are internalizing nanoparticles. Following staining, lymph node
cell suspensions are analyzed
by flow cytometry (CyAn ADP, Dako, Glostrup, Denmark). Further analysis is
performed using FlowJo
software (TreeStar, Ashland, Oreg.). APCs and DCs with internalized
fluorescent nanoparticles are
determined to be MHCII+FITC+ and CD11c+FITC+, respectively, FITC representing
labeling of the
nanoparticles. DC maturation following nanoparticle internalization is
evaluated by calculating the
fraction of cells that expressed CD86 and CD80.
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Example 9. Modification of Quantum Dot Nanoparticle
[00162] The amine surface of the quantum dot nanoparticle including dendrimer-
encapsulated
quantum dots is modified with a linking agent such as Sulfo-SMCC
(Sulfosuccinimidyl 4-N-
maleimidomethyl cyclohexane-1-carboxylate) to form a maleimide-activated
quantum dot. Any excess
reagent is removed by size-exclusion chromatography, a commonly-employed
technique to separate the
components of the reaction mixture. Methods for encapsulating quantum dots
with dendrimers are
described in, for example, Lemon et al. Q. Am. Chem. Soc., 2000, 122:12886),
the contents of which are
incorporated herein by reference.
[00163] Formation of Quantum Dot Nanoparticle-AChE Conjugate
[00164] Maleimide-activated QD is reacted with the sulfhydryl-modified AChE
for a limited time to
prevent multiple QDs from attaching to multiple proteins. Multiple cross
linking is not favored due to
steric hinderance and entropy of large molecules. The reaction is halted by
separation using size-exclusion
chromatography.
Example 10. Method of producing dendrimers
[00165] PAMAM dendrimers are composed of an ethylenediamine (EDA) initiator
core with four
radiating dendron arms, and are synthesized using repetitive reaction
sequences comprised of exhaustive
Michael addition of methyl acrylate (MA) and condensation (amidation) of the
resulting ester with large
excesses of EDA to produce each successive generation. Each successive
reaction therefore theoretically
doubles the number of surface amino groups, which can be activated for
functionalization. The
synthesized dendrimer has been analyzed and the molecular weight has been
found to be 26,380 g/mol by
GPC and the average number of primary amino groups has been determined by
potentiometric titration to
be 110.
Example 11. Characterization of Dendrimer Functional Groups
[00166] Acetylation of the dendrimer.
[00167] Acetylation is the first requisite step in the synthesis of
dendrimers. Partial acetylation is used
to neutralize a fraction of the dendrimer surface from further reaction or
intermolecular interaction within
the biological system, therefore preventing non-specific interactions from
occurring during synthesis.
Leaving a fraction of the surface amines non-acetylated allows for attachment
of functional groups.
Acetylation of the remaining amino groups results in increased water
solubility (after FITC conjugation),
allowing the dendrimer to disperse more freely within aqueous media with
increased targeting specificity,
as compared to many conventional mediums (Quintana et al., Pharm. Res. 19,
1310 (2002)).
Example 12. Conjugation of Functional Groups to Acetylated Dendrimer
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[00168] Conjugation of fluorescein isothiocyanate to acetylated dendrimer. A
partially acetylated
PAMAM dendrimer is used for the conjugation of fluorescein isothiocyanate
(FITC) in order to increase
the solubility of the dendrimer. The partially acetylated dendrimer is allowed
to react with fluorescein
isothiocyanate, and after intensive dialysis, lyophilization and repeated
membrane filtration the
dendrimer-FITC product is yielded.
[00169] Conjugation of folic acid to acetylated dendrimer.
[00170] Conjugation of folic acid to the partially acetylated mono-functional
dendritic device is
carried out via condensation between the y-carboxyl group of folic acid and
the primary amino groups of
the dendrimer. This reaction mixture is added drop wise to a solution of DI
water containing dendrimer-
FITC and vigorously stirred for 2 days (under nitrogen atmosphere) to allow
for the folic acid to fully
conjugate to the dendrimer-FITC.
Example 13. Evaluating Tolerance by T cell Phenotypic Analysis
[00171] A nanoparticle-MBP(83-99) complex of the invention is dissolved in
phosphate-buffered
saline (PBS) and injected into Female Lewis rats intraperitoneally in 0.1-0.2
ml containing 500 gg of the
nanoparticle-MBP complex. The control group of rats receives 0.1-0.2 ml of
PBS. Nine to ten days after
the injection, spleen and lymph nodes (inguinal and lumbar) are harvested from
the rats and single cell
suspensions obtained by macerating tissues through a 40 m nylon cell
strainer. Samples are stained in
PB S (1 % FC S) with the appropriate dilution of the relevant monoclonal
antibodies. Propidium iodide
staining cells are excluded from analysis. Samples are acquired on an LSR2
flow cytometer (BD
Biosciences, USA) and analysed using FACS Diva software. The expression of the
activation markers
CD25, CD44, CD62L, CTLA-4, CD45Rb, and CD69 is analyzed on splenocytes and
lymph node cells
from the rats injected with the nanoparticle-MBP complex. The CD4+ T cells
from the complex-injected
rats express a CD25h` / CD45RB''t phenotype, which is characteristic of
anergized CD4+ T cells. There is
also a higher percentage of CD4+ cells that is CD25h' and FoxP3+, suggesting
the induction of regulatory T
cells.
[00172] To assess apoptosis, CD8+ T cells are isolated from splenocytes using
a CD8a isolation kit
(Miltenyi Biotec, Germany) according to the manufacturer's protocol, stained
with annexin V and PI (both
BD Biosciences) according to the manufacture's protocol and then analyzed by
flow cytometry. For
intracellular staining of Granzyme B and Bcl-2, T cells are permeabilized with
BD cytofix/cytoperm kit
(BD Biosciences) and stained with rat-anti-mouse Granzyme B PE mAb
(eBioscience) or hamster-anti-
mouse Bcl-2 FITC mAb (BD Biosciences). Specificity controls are performed
using the appropriate
isotype mAbs. Samples are analyzed by flow cytometry, which is well known in
the art.
Example 14. Evaluating Tolerance by T cell Proliferation
[00173] A nanoparticle-MBP(83-99) complex of the invention is dissolved in
phosphate-buffered
saline (PBS) and injected into Balb/c mice intraperitoneally in 0.1-0.2 ml
containing 500 g of the
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nanoparticle-MBP complex. The control group of mice receives 0.1-0.2 ml of
PBS. Nine to ten days after
the injection, spleen and lymph nodes (inguinal and lumbar) are harvested from
the mice and single cell
suspensions obtained by macerating tissues through a 40 tm nylon cell
strainer. CD4+ T cells are isolated
from splenocytes using a CD4+ T cell isolation kit (Miltenyi Biotec, Germany)
according to the
manufacturer's protocol. Purified CD4+ T cells are plated (5 x 104/well) in
triplicate with 1) T cell-
depleted, irradiated (2000 R) CBA/J stimulators (5 x 105) for 72 hr, 2)
syngeneic splenocytes (5 x 105)
plus soluble anti-CD3 (145-2C11) for 48 hr, or 3) 100 ng/ml phorbol 12-
myristate 13-acetate (Sigma) and
200 nM calcium ionophore (ionomycin, Sigma) for 36 hr total, with a pulse of 1
p.Ci/well [3H]TdR during
the last 8-12 hr. [3H]TdR incorporation as an indicator of DNA replication and
cell proliferation is
measured in the presence of scintillation fluid on a [i-counter (Beckman
Coulter, Inc., Fullerton, CA).
Supernatants from anti-CD3-stimulated T cells are harvested at 48 hr, and the
production of 1L-2 is
determined by measuring proliferation of the IL-2-dependent cell line CTLL-2
in the presence of anti-IL-4
antibody (I1B11), where 1 unit is the amount of IL-2 required to support half-
maximal [3H]TdR
incorporation.
[00174] T cell proliferation may also be assessed by directly visualizing the
division of cells using the
fluorescent cytosolic dye, Carboxyfluorescein Diacetate Succinimidyl Ester
(CFSE). For CSFE labeling
of cells, splencoytes from the mice injected with the nanoparticle-MBP complex
are incubated with 3 M
CFSE (Molecular Probes, UK) for 3 min at 37 C in Iml of PBS. In each well of a
96 well-plate, 2x 105
CF SE-labeled splenocytes are stimulated with 1 x 105 irradiated (80 Gy)
temperature-induced RMA-S
coated with MBP(8 3-99) peptide (10 M) or an irrelevant control peptide, pSV9
(10 M). The
appropriate cultures are supplemented with 10 U/ml IL-2, 10 ng/ml IL-7, 50
ng/ml IL-15 or 50 ng/ml IL-
21. Cells are harvested at the appropriate time point, stained with CD4 and
subjected to CFSE profiling by
flow cytometry.
Example 15. Evaluating Tolerance by T cell Cytokine Profile and Cytotoxicity
IFN-y assays
[00175] A nanoparticle-MBP(83-99) complex of the invention is dissolved in
phosphate-buffered
saline (PBS) and injected into Balb/c mice intraperitoneally in 0.1-0.2 ml
containing 500 g of the
nanoparticle-MBP complex. The control group of mice receives 0.1-0.2 ml of
PBS. Nine to ten days after
the injection, spleen and lymph nodes (inguinal and lumbar) are harvested from
the mice and single cell
suspensions obtained by macerating tissues through a 40 tm nylon cell
strainer. To measure antigen
specific IFN-y production, CD4+ T cells are isolated from splenocytes using a
CD4+ T cell isolation kit
(Miltenyi Biotec, Germany) according to the manufacturer's protocol. Purified
CD4+ T cells are
stimulated, culture supernatant harvested and IFN-y measured in an IFN-y
ELISA. Triplicate cultures are
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plated for each different experimental condition on 96-well round-bottom
plates. In each well 1 X 104
splenocytes from the mice injected with the nanoparticle-MBP(83-99) complex
are stimulated with 1 X 104
irradiated (80 Gy) temperature-induced RMA-S coated with MBP(83-99) peptide
(10 M) or an irrelevant
control peptide, pSV9. The appropriate cultures are supplemented with 10 U/ml
IL-2, 10 ng/ml IL-7, 50
ng/ml IL-15 or 50 ng/ml IL-21. After 72 hours, 50 l culture supernatant is
harvested from each well and
murine IFN-y is measured by sandwich ELISA using anti-IFN-y antibodies (BD
Biosciences). The
activity in experimental samples is ascertained using the standard curve of
mean absorbance values (OD)
versus the dilution of recombinant IFN-y in the supernatant.
IL-2 bioassays
[00176] To measure antigen specific IL-2 production, purified CD4+ T cells
from the mice injected
with the nanoparticle-MBP(83-99) complex are stimulated, culture supernatant
harvested and IL-2
measured using IL-2 dependent CTLL cells. The stimulation phase of the IL-2
assay is performed exactly
as the IFN-y assay. After 72 hours, 50 l culture supernatant is harvested,
transferred to wells containing
CTLL cells (5x 103) and incubated for 16-18 hours. The cells are pulsed with
3[H]-thymidine by adding 1
Ci 3 [H]-thymidine to each well and incubating for a further 12 hours. The
activity in experimental
samples is ascertained using the standard curve of cpm versus the dilution of
recombinant IL-2 in the
supernatant. Alternatively, murine IL-2 from the supernatant may be measured
by sandwich ELISA using
anti-IL-2 antibodies (BD Biosciences).
CTL assays
[00177] Cytotoxic activity of CD8+ T cells from the mice injected with the
nanoparticle-MBP(83-99)
complex is determined in 4-hour "chromium-release assays against MBL-2 tumor
cells and RMA-S cells
coated with MBP(83-99) peptides or MHC class I-binding control peptides.
51chromium-release assays are
well known in the art.
Cytokine ELISA
[00178] Purified CD4+ T cells from the mice injected with the nanoparticle-
MBP(83-99) complex are
stimulated and the culture supernatant is harvested as described hereinabove.
Levels of cytokines
including IL-1, IL-4, IL-5, IL-6, IL-10, IL-13, TGF-fl, and TNF-a are measured
by cytokine sandwich
ELISA using the standard protocols (BD Biosciences). Increased production of
IL-4, IL-5, IL- 10 and IL-
13 is typically associated with a Th2 response. IL-10 and TGF-f3 are typically
associated with a regulatory
T cell response.
Example 16. Evaluating Tolerance by T cell Suppressive Activity
1001791 A nanoparticle-MBP(83-99) complex of the invention is dissolved in
phosphate-buffered
saline (PBS) and injected into Balb/c mice intraperitoneally in 0.1-0.2 ml
containing 500 g of the
nanoparticle-MBP complex. The control group of mice receives 0.1-0.2 ml of
PBS. Nine to ten days after
the injection, spleen and lymph nodes (inguinal and lumbar) are harvested from
the mice and single cell
suspensions obtained by macerating tissues through a 40 p.m nylon cell
strainer. To measure T cell
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suppressive activity, CD4+ T cells are isolated from splenocytes using a CD4+
T cell isolation kit
(Miltenyi Biotec, Germany) according to the manufacturer's protocol. CD4+CD25+
regulatory T cells can
be isolated using CD25 microbeads.
[001801 Culture the CD4+CD25- responder T cells (Tresp) (3 x 104) in U-bottom
96-well plates with
Treg (CD4+CD25+) from the mice injected with the nanoparticle-MBP(83-99)
complex in the presence of
soluble 0.5-0.75 tg/ml a-CD3 and 2.5 - 4 ug/ml a-CD28 for 2-4 days. 3x 104
irradiated (3000 Rad) T cell-
depleted splenocytes can be added as APCs instead of a-CD28 in the co-culture.
Label Tresp cells with
CFSE to distinguish them from the Treg cells in the co-culture. Measure
proliferation of the CD4+CD25-
responder T cells by incorporation of 3H- thymidine for the last 6-16 h of
culture or by CFSE dilution.
Measure cell death at 0 hr and after 1, 2 and 3 days. Cell death analyses of
CFSE+ responders should be
performed based on forward scatter or Annexin V and propidium iodide staining.
Treg isolated from the
mice injected with the nanoparticle-MBP(83-99) complex are capable of
suppressing the proliferation of
the responding CD4+CD25- T cells.
Example 17. Induction of Tolerance With gp39 Peptide In Vivo
[001811 A human cartilage (HC) gp-39 (263-275)-specific delayed type
hypersensitivity (DTH) assay
suitable to monitor tolerance induction with peptide antigens is developed.
Immunization of Balb/c mice
with HC gp-39 (263-275) in incomplete Freunds adjuvant (IFA) is effective in
the induction of a DTH
response following challenge with the HC gp-39 (263-275) peptide. This peptide-
based DTH system is
used to detect modulation of the DTH response by parenteral application of
nanoparticles conjugated with
HC gp-39 (263-275) peptide. Application of the nanoparticles conjugated with
HC gp-39 (263-275)
peptide, in a dose-dependent manner, downmodulates the HC gp-39 (263-275)
induced DTH response,
indicating that the nanoparticles conjugated with HC gp-39 (263-275) peptide
can efficiently tolerize a
peptide-specific response induced with HC gp-39 (263-275).
Example 18. Induction of Tolerance in a HLA:DR2 Transgenic Mouse
[00182] An antigen-carrier complex of the present invention, when presented by
an MHC molecule,
can induce immunological tolerance in a humanized mouse model of multiple
sclerosis using a peptide
selected from within T-cell epitopes of myelin basic protein (MBP)
corresponding to MBP 140-154. The
mouse model is transgenic for the human MHC molecule HLA:DR2 (DRB1 * 1501)
(Madsen et at. (1999)
Nature Genetics 23:343-347).
[00183] The induction of anergy or changes in the CD4+ T-cell population in a
mouse following
administration of an antigen-carrier complex may be monitored by a reduction
in T-cell proliferation
when challenged with the antigen in vivo.
Methods
Antigens
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[00184] MBP peptide 140-154 is synthesized using L-amino acids and standard F-
moc chemistry on
an Abimed AMS 422 multiple peptide synthesizer (Abimed, Langenfeld, Germany).
The sequence of
MBP peptide 140-154 is GFKGVDAQGTLSKIF. MBP peptide 140-154 is conjugated to
the
nanoparticles as described herein. Purified Protein Derivative of
Mycobacterium tuberculosis (PPD)
(Veterinary Laboratories, Addlestone, Surrey) is used at a concentration of 50
tg/m1 in lymphocyte
proliferation assays.
Mice and Tolerance Induction
[00185] HLA:DR2 transgenic are bred in isolators and housed in a specific
pathogen-free facility.
Within each treatment group, mice are both age (8-12 weeks) and sex matched.
Mice are pre-treated with
100 ug of MBP peptide 140-154 in 25 ul of phosphate buffered saline (PBS) or
25 ul PBS alone
intranasally (i.n) on days -8, -6 and -4 prior to immunization on day 0.
[00186] Mice are immunized subcutaneously at the base of the tail and hind
limb with 100 ul of an
emulsion consisting of an equal volume of Complete Freund's Adjuvant (CFA) and
PBS containing 200
ug MBP140-154 and 400 ug heat-killed Mycobacterium tuberculosis, strain
H37RA(Difco, Detroit,
Mich.). A control group of mice, previously treated with PBS i.n., are
immunized without peptide.
[00187] Intranasal pre-treatment followed by immunization give rise to three
groups of mice: Group A
is tolerized with PBS and immunized with MBP 140-154 (7 mice); Group B is
tolerized with MBP 140-
154-nanoparticles and then immunized with the same peptide MBP 140-154 (7
mice); and Group C is
both tolerized and immunized with PBS.
Lymph Node Proliferation Assays
[00188] After 10 days, draining popliteal and inguinal lymph nodes are removed
aseptically. The
nodes are disaggregated, washed and resuspended in X-Vivo 15 medium
(BioWhittaker, Maidenhead,
UK) supplemented with 5x 10-5 M 2-mercaptoethanol and 4 mM L-glutamine. Cells
are plated in triplicate
at 5x105 cells well and cultured with or without varying concentrations of MBP
peptide 140-154 (1-150
ug/ml) for 72 hours. To check for successful immunization of mice, lymph node
cells are plated with PPD
(50 ug/ml) as described above. Cultures are pulsed for the final 16 hours with
0.5 uCi [3H]-Thymidine.
Cells are harvested and T-cell proliferation is expressed as Stimulation Index
(SI): corrected counts per
minute (ccpm) antigen containing culture/cepm culture without antigen.
Results
[00189] Mice that are pre-treated i.n. with PBS and then immunized with MBP
peptide 140-154
(Group A) respond to antigenic stimulation when re-challenged with MBP140-154
in a dose dependent
manner. With increasing concentration of peptide, the SI, a measure of
lymphocyte proliferation,
increases from a median of 2.5 to 10. All the mice in this group demonstrate
that PBS administered
intranasally could not induce tolerence to MBP 140-154. In contrast,
intranasal pre-treatment with MBP
140-154-nanoparticle have a profound effect on the proliferative response of
lymphocytes stimulated with
this peptide. Lymphocytes from Group B mice are unable to respond to any
significant degree, even at the
high peptide concentration of 150 ug/ml (SI median 3). The marked reduction in
proliferation in Group B
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as compared with Group A is evident. The data shows that the MBP 140-154-
nanoparticles of the present
invention induce tolerance in lymphocytes from HLA-DR2 mice.
[00190] Lymphocytes extracted from mice which have been pre-treated and
immunized with PBS
(Group C) fail to show any response to MBP 140-154 suggests, although they
elicit an excellent response
to PPD and are therefore immunized against the PPD antigen, This lack of
response to MBP peptide
within Group C confirms that the proliferative response seen in Group A is
indeed a response to
immunization with MBP 140-154, and that the responses to both MBP 140-154 and
PPD in control
Groups A and C are antigen specific. As the proliferation to MBP 140-154 is
antigen specific, induction
of tolerance to this molecule is also specific.
CONCLUSION
[00191] An MBP peptide-nanoparticle of the invention, e.g., MBP 140-154, that
does not require
processing and binds to HLA:DR2 MHC Class II molecules, can induce tolerance
when administered
intranasally.
Example 19. Treatment of EAE with the Antigen-Carrier Complex
Immunizations and EAE Induction
[00192] MBP peptide and peptide analogs are dissolved in phosphate-buffered
saline (PBS) and
emulsified with an equal volume of incomplete Freund's adjuvant supplemented
with 4 mg/ml heat-killed
Mycobacterium tuberculosis H37Ra in oil (Difco Laboratories, Inc., Detroit,
Mich.). Female Lewis rats
are immunized subcutaneously at the base of the tail with 0.1-0.2 ml
containing 500 ug of peptide in the
emulsion and are monitored for clinical signs daily. EAE is scored on a scale
of 0-4, as follows: 0,
clinically normal; 1, flaccid tail; 2, hind limb weakness; 3, hind limb
paralysis; 4, front and hind limbs
affected.
[00193] In this system, experimental allergic encephalomyelitis (EAE) is
induced in twelve female
Lewis rats by injection of MBP(83-99) peptide in complete Freund's adjuvant
(CFA) at the base of the
tail. Nine days later, rats are divided into two groups of six animals and
subcutaneously injected with 13.2
mg/kg of either MBP peptide-dendrimer or a control peptide, sperm whale
myoglobin (SWM) (110-121).
Animals are monitored daily for disease symptoms and scored in a blinded
fashion on a nonlinear
ascending scale of 0-4 with increments denoting increasing paralysis. Each
individual score is averaged
with group cohorts to obtain the mean clinical score.
[00194] The disease severity in those animals treated with the MBP peptide-
dendrimer is about 50%
reduced compared to the control group. The MBP peptide-dendrimer reduces the
severity and duration of
the disease in this model system.
[00195] Although these results clearly demonstrate that MBP peptide-dendrimer
inhibits the
development of EAE, a murine animal model system of EAE has also been
developed. The SJL/J (H-25)
mouse develops a chronic relapsing form of EAE in response to immunization
with MBP(83-99) peptide
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in the presence of pertussis vaccine. The ability of the MBP peptide (83-99)-
dendrimer to inhibit the
disease is evaluated.
[00196] Groups of 10 animals are injected intraperitoneally weekly for 4 weeks
with 20 mg/kg of
either a control peptide or the peptide analog. The animals are then monitored
for disease over the next 2-
3 months. SJL/J mice develop symptoms of EAE beginning around day 20 in the
control group that last
for approximately 3 weeks. Beginning around day 70, a relapse occurs reaching
a mean clinical score of
about 1. However, weekly injection with the MBP peptide (83-99)-dendrimer for
four weeks not only
reduces the level of the disease in the first phase, but also reduces the
severity of the relapse.
Example 20. Production and use of peptide-coupled polystyrene microspheres
Production of peptide-coupled polystyrene microspheres
[00197] If necessary, warm the carboxyl microparticles, PolyLink Coupling
Buffer and PolyLink
Wash/Storage Buffer (Polysciences, Inc., Warrington, PA) to room temperature.
Carboxyl (COOH)
microparticles can be used for covalent coupling of proteins by activating the
carboxyl groups with water-
soluble carbodiimide (ECDI). The carbodiimide reacts with the carboxyl group
to create an active ester
that is reactive toward primary amines on the protein of interest. Pipet
12.5mg of microparticles into a 1.5
polypropylene microcentrifuge tube. Pellet the microparticles via
centrifugation for 5-10 minutes at
approximately 10000 x G. Resuspend the microparticle pellet in 0.4m1 of
PolyLink Coupling Buffer.
Pellet again via centrifugation for 5-10 minutes at approximately 10000 x G.
Resuspend the microparticle
pellet in 0.17m1 of PolyLink Coupling Buffer. Just before use, prepare a
200mg/ml ECDI solution by
dissolving 10mg PolyLink ECDI in 50 l PolyLink Coupling Buffer. Add 201l of
the ECDI solution to the
microparticle suspension. Mix gently end-over-end or briefly vortex. Add
protein equivalent (e.g.
PLP139-151, PLP179-191 or OVA323_339) to 200-500 g. Mix gently by pipetting.
Incubate for 30-60 minutes at
room temperature. Centrifuge mixture for 10 minutes at approximately 10000 x
G. Save this supernatant
for determination of the amount of bound protein. Resuspend microparticle
pellet in 1mL sterile PBS.
Centrifuge again at 10000 x G.
Inj ection of peptide-coupled polystyrene microspheres
[00198] Resuspend in 1mL of sterile PBS. Pass suspension across a 40 m mesh
strainer to remove
clumps of cross-linked particles. Raise volume to 4mL with sterile PBS
(500micrograms of coupled
microspheres is sufficient to dose 20 animals). Inject suspended particles
into mice via the lateral tail
vein.
Example 21. Peptide-coupled polystyrene microspheres induce specific tolerance
for both prevention and
treatment of PLP-induced EAE
[00199] This example describes the effect of administration of peptide-coupled
polystyrene
microspheres either prior to, or after induction of PLP139-151 induced EAE in
mice.
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WSGR Docket No. 28890-717.601
[002001 The production of peptide coupled microspheres was performed either as
described in
Example 1 or Example 20. Either PLP139-151 or a control (OVA323_339) peptide
was coupled to 0.5 m
microspheres. Mice were injected intravenously with either the PLP139-151 or
control (OVA323_339) peptide
bound microspheres either on day -7 ("Disease Prevention) or day 12 ("Disease
Treatment") relative to
priming with PLP139_151 or PLP178_191 + Complete Freund's Adjuvant (CFA) on
day 0. Animals were
observed and scored as described in Example 1. The results are shown in Figure
2. Animals treated prior
to disease onset with PLP139-151-coated microspheres showed a decrease in
clinical score compared to
animals treated with Sham beads (microspheres treated with ECDI but no
peptide). The results also
showed a similar decrease in clinical score to treatment using cells treated
to have PLP139-151 on the cell
surface (see FIG 2A and 2B). Animals treated following disease onset with PLPI
39-151 -coated
microspheres similarly showed a decrease in clinical score compared to animals
who were either untreated
or treated with microspheres having a control peptide (see FIG 2C). Therefore,
the results show that
treatment using peptide-coupled polystyrene microspheres is useful for
decreasing disease severity prior
to and following disease onset.
Example 22. Recall responses to primed and spread epitopes are decreased in
tolerized recipients
[00201] This example describes the effect of administration of peptide-coupled
polystyrene
microspheres on delayed type-hypersensitivity in a mouse model.
[00202] Mice were prepared and treated using either PLP139-151, control
(OVA323-339) peptide, or sham
bound microspheres. Recall responses of CD4 T-cells were measured by delayed-
type hypersensitivity
(DTI) at day 40 relative to priming with PLP139_151 or PLP178-19 1/Complete
Freund's Adjuvant (CFA) on
day 0, as described previously (Smith and Miller (2006) Journal of
Autoimmunity 27:218-31; Luo et al.
(2008) PNAS 105:14527-32). Measurement of DTH was done by measurement of ear
swelling using a 24
hour ear swilling assay. Pre-challenge ear thickness was determined using a
Mitutoyo model 7326
engineer's micrometer (Schlesinger's Tools, Brooklyn, NY). Immediately
thereafter, DTH responses
were elicited by injecting peptide into the dorsal surface of the ear . The
increase in ear thickness over
pre-challenge measurements was determined 24 hours after ear challenge.
Results are shown in FIG. 3.
The mean net swelling in mice pre-treated with PLP139-151 microspheres was
comparable to control,
whereas the control (OVA323_339) peptide, or sham bound microspheres resulted
in increased swelling.
These results indicate that microparticles can be used to protect animals from
later inflammatory
responses.
Example 23. Effect of peptide-coupled microspheres on CNS infiltration
[002031 This example describes the effect of administration of peptide-coupled
polystyrene
microspheres on CNS infiltration of leukocytes into the CNS.
[00204] Mice were prepared and treated using either PLP139-151, control
(OVA323-339) peptide bound
microspheres or were not treated with any microspheres. Mice were injected on
day -7 relative to
46
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WSGR Docket No. 28890-717.601
priming with PLP139_151/Complete Freund's Adjuvant (CFA). Mouse infiltration
of leukocytes into the
CNS were examined by immunohistochemistry, as described previously (Smith and
Miller (2006) Journal
of Autoimmunity 27:218-31; Turley and Miller (2007) J Immunol. 178:2212-20).
Briefly, mice were
anesthetized and perfused with 30m1 PBS on the indicated days post-
immunization. Spinal cords were
removed by dissection and immediately frozen in liquid nitrogen. Six
micrometer thick cross-sections
from the lumbar region were cut and mounted on Superfrost Plus
electrostatically charged slides (Fisher,
Pittsburgh, PA), air dried, and stored at -80 C. The results are shown in FIG.
4. Slides were stained for
cellularity (FIG. 4A), CD4+CD3+ cells (FIG. 4B) or Foxp3+ cells (FIG.4C) in
the CNS. The results
indicate that treatment with PLP139-151 treated microspheres resulted in a
decrease in leukocytes in the
CNS compared to control.
Example 24. Effective of peptide coupled microspheres on splenectomized
animals
[00205] This example describes the effect of administration of peptide-coupled
polystyrene
microspheres on mice that were splenectomized to examine the requirement of
spleen activity in the
induction of tolerance.
[00206] The production of peptide coupled microspheres was performed either as
described in
Example 1 or Example 20. Splenectomized or intact animals were treated with
PLP139.151, control
(OVA323_339) peptide bound microspheres or were not treated with any
microspheres. The animals were
treated with microspheres on day -7 relative to priming with
PLP139_151/Complete Freund's Adjuvant
(CFA). The results are shown in FIG. 5. The control (OVA323-339) peptide bound
microspheres showed a
mean clinical score similar to animals that were not treated with any
microspheres, whereas both mice
splenectomized or intact that were treated with PLP139-151 microspheres showed
a decrease in mean
clinical score.
47
